Rubus Fruticosus and Verbascum Thapsus Medicinal Plants Collected from Dir (L) N.W.F.P

Antioxidant and anti-inflammatory study of

Rubus fruticosus and Verbascum thapsus medicinal plants collected from Dir (L) N.W.F.P.

Pakistan

Muhammad Riaz, B. Pharm.

Department of Pharmacognosy

Faculty of Pharmacy, University of Karachi

Karachi-75270, Pakistan

2012 Antioxidant and anti-inflammatory study of

Rubus fruticosus and Verbascum thapsus medicinal plants collected from Dir (L) N.W.F.P.

Pakistan

THESIS SUBMITTED FOR THE FULFILMENT

OF THE DEGREE OF

DOCTOR OF PHILLOSOPHY

Muhammad Riaz, B. Pharm.

Supervised by

Dr. Mansoor Ahmad, I.F.

Meritorious Professor

Department of Pharmacognosy

Faculty of Pharmacy, University of Karachi

Karachi-75270, Pakistan

2012

DIDICATED TO MY PARENTS

PUBLICATION FROM THESIS Riaz M , Ahmad M and Rahman N (2011). Antimicrobial screening of fruit, leaves, root and stem of Rubus fruticosus . J. Med. Plants Res ., 5(24): 5920-5924.

CONTENTS i. Acknowledgements viii ii. Abstract ix iii. Khulasa xii 1. Introduction 01 I. Rubus fruticosus 03 II. Verbascum thapsus 07 2. Literature search i. Literature survey of Rubus fruticosus 13 ii. Therapeutic application of Rubus fruticosus 15 iii. Literature data for total phenols, anthocyanins and ascorbic acid 17 iv. Phytochemical literature survey of Rubus fruticosus 18 v. Structures of chemical constituents reported from R. fruticosus 23 vi. Literature survey of Verbascum thapsus 28 vii. Pharmacological literature survey of Verbascum thapsus 30 viii. Phytochemical literature survey of Verbascum thapsus 32 ix. Structures of chemical constituents reported from V. thapsus 38 3. Experimental 42 i. General/Materials 42 ii. Instruments 43 iii. Abbreviations 44 iv. Pharmacognostic evaluation/Standardization of drugs 45 v. Thin layer chromatography 48 vi. Pharmacological Analysis 48 vii. Anti-oxidant activity 48 viii. Anti-inflammatory activity 49 ix. Analgesic activity 50 x. Diuretic activity 51 xi. Gross behavioural activity 52 xii. Neuropharmacological Activity 54

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xiii. Antimicrobial activity 56 xiv. Toxicity Studies 59 4. Results 60 5. Discussion 221 6. Conclusion 236 7. References 237

LIST OF TABLES ******************** Table 1a and 1b : An overview of chemical constituents identified by different chemical reagents 72 Table 2a and 2b : An overview of chemical constituents identified by different chemical reagents 74 Table 3 - 5: Fluorescence analysis of powder of Rubus fruticosus 76 Table 6 - 8: Fluorescence analysis of powder of Verbascum thapsus 77 Table 9: Fluorescence analysis of extracts of Rubus fruticosus 79 Table 10 : Fluorescence analysis of extracts of Verbascum thapsus 80 Table 11 : Rf values of Rubus fruticosus extracts by Thin layer chromatography 81 Table 12 : Rf values of Rubus fruticosus extracts by Thin layer chromatography 82 Table 13 and 14: Antioxidant activity of Rubus fruticosus and Verbascum thapsus extracts using DPPH assay 83 Table 15 and 16: Antioxidant activity of Rubus fruticosus and Verbascum thapsus extracts using ABTS and Nitric oxide assay 84 Table 17: Assessment of anti-inflammatory activity (Formalin induced inflammation) 85 Table 18: Assessment of anti-inflammatory activity (Carrageenan 86 induced inflammation) Table 19 and 20 : Effect of crude extract of on Hot plate Analgesiometer 87 in mice Table 21: Assessment of analgesic activity (Acetic acid 89 induced writhing) Table 22 and 23 : Effect of crude extract of on water bath (tail flick) in mice 90 Table 24: Diuretic activity of Rubus fruticosus and Verbascum thapsus (Various parts extracts) 92 Table 25-27: Behavioural response of Rubus fruticosus (Fruit) (100, 300, 500) in mice 93 Table 28- 30 : Behavioural response of Rubus fruticosus (Leaves) (100, 300, 500) in mice 96 Table 31- 33: Behavioural response of Rubus fruticosus (Root) (100, 300, 500) in mice 99 Table 34- 36: Behavioural response of Rubus fruticosus (Stem) (100, 300, 500) in mice 102 Table 37- 39: Behavioural response of Verbascum thapsus (Fruit) (100, 300, 500) in mice 105 Table 40- 42 : Behavioural response of Verbascum thapsus (Leaves) (100, 300, 500) in mice 108 Table 43- 45: Behavioural response of Verbascum thapsus (Root) (100, 300, 500) in mice 111 Table 46- 48: Behavioural response of Verbascum thapsus (Stem) (100, 300, 500) in mice 114 Table 49 and 50 : Assessment of neuropharmacological activity in

30 minutes (open field and head dip activity) 117 Table 51 and 52 : Assessment of Exploratory activity (Rearing & cage cross) 119 Table 53 and 54: Assessment of Neuropharmacological activity (traction time) 121 Table 55 and 56: Assessment of Forced swimming activity 123 Table 57-64: Antibacterial activity of Rubus fruticosus and Verbascum thapsus (Fruit, leaves, root and stem extract) 125 Table 65: Antifungal activity of Rubus fruticosus and Verbascum thapsus (Fruit, leaves, root and stem extract) 129 Table 66-73: Insecticidal activity of standard drug, Rubus fruticosus and Verbascum thapsus (Fruit, leaves, root and stem) 129 Table 74-82: Anthelmintic activity of standard drug, Rubus fruticosus and Verbascum thapsus (Fruit, leaves, root and stem) 134 Table 83-91: Brine shrimp lethality test of Rubus fruticosus , Verbascum thapsus and standard 143

LIST OF GRAPHS *********************

Graph 1 : Anti-oxidant activity determination of Rubus fruticosus, Verbascum thapsu s 148 Graph 2: Anti-inflammatory activity (Formalin induced inflam- 152 mation) of Rubus fruticosus and Verbascum thapsu s Graph 3: Anti-inflammatory activity (Carrageenan induced inflam- 156 mation) of Rubus fruticosus and Verbascum thapsus Graph 4: The effect of hot plate activity of Rubus fruticosus and Verbascum thapsu s 160 Graph 5: Analgesic activity of Rubus fruticosus and Verbascum thapsu s by acetic acid induced writhing test 164 Graph 6: Analgesic activity of Rubus fruticosus and Verbascum 168 thapsu s through tail flick water bath Graph 7: Open field and head dip activity of Rubus fruticosus 172 and Verbascum thapsus Graph 8: Cage cross and rearing activity of Rubus fruticosus 176 and Verbascum thapsus Graph 9: Effect of Rubus fruticosus and Verbascum thapsu s 180 on mobility time Graph 10: Antibacterial activity of Rubus fruticosus and 184 Verbascum thapsus Graph 11: Brine shrimp lethality test of Rubus fruticosus, 216 Verbascum thapsus and standard

LIST OF FIGURES ********************** Figure 1: TLC finger prints of Rubus fruticosus, Verbascum thapsus 146

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ACKNOWLEDGEMENTS

I would like to express my eternal gratitude to my thesis supervisor Prof. Dr. Mansoor Ahmad for his constant and strong knowledge, inspiration and indefinite kindness. Without his continuous inspiration this work would not have been accomplished at this moment. He shall always remain an unstinting inspiration for me, for the rest of my life. His valuable suggestions as final words during the research work are greatly acknowledged. His kind thoughts and wishes have been tremendous comforts to my whole research work.

I am also thankful to the respected Chairman, Department of Pharmacognosy, Prof. Dr. Iqbal Azhar and the respected Dean Faculty of Pharmacy, Prof. Dr. Ghazala Hafeez Rizwani . I offer my heartiest gratitude to all teaching faculty for their kindness, help and support, which made my stay in department of Pharmacy, University of Karachi, and a momentous period of my life.

I am also grateful to Higher Education Commission Islamabad, Pakistan for financial support. I also offer my sincere gratitude to Dr. Mehjabeen and Dr. Noor Jahan , who being my seniors helped me in conducting pharmacological part of my research work. I will never forget inspiration and kind attitude Mr. Zahid Khan to me in lab. I heartily acknowledge their kindness and support. I would also thankful to the lab staff of the Department of Pharmacognosy, especially Mr. Noman-ul-haq and Tauseef Ahmed for their help during work. My sincere thanks are due to my colleagues and friends, Mr. Najam Ur Rehman and Mr. Rizwan Ahmad for providing me enormous support during the whole time. I owe special thanks to them for completing this assignment.

Last, but not the least my Parents and Family that with their continuous support outlined my academic career. Their prayers and love made many of my academic dreams turning into glaring realities. Without their love and support this work would have been a distant point in my life. Muhammad Riaz viii

ABSTRACT The aim of this study is not only to preserve the traditional information but also to update and expand this knowledge of medicinal plants according to modern parameters. In this regard two species Rubus fruticosus (Rosaceae) and Verbascum thapsus (Scrophulariaceae) were collected from Northern area Dir (KPK formally called NWFP) of Pakistan which is very rich in medicinal plants, not only to evaluate but to validate their traditional uses according to advanced screening techniques. Methanolic extract of each part of both species was investigated and evaluated for anti-inflammatory, antioxidant potentials, pharmacognostic, neuropharmacological, gross behavioral studies, toxicity studies and diuretic action.

Pharmacognostic studies The presence of carbohydrates, triterpenes, tannins, flavonoids and sterols were detected in R. fruticosus and V. thapsus during chemical screening. Thin layer chromatographic plates were also developed for the extract of each part of the plant. The Rf value of each spot was calculated during experiment, for R. fruticosus fruit (0.38,0.42,0.59,0.66,0.73,0.81) for leaves (0.33,0.62,0.64,0.72,0.75) for root (0.38, 0.43) and for stem extract (0.36, 0.43) in Ethyl acetate, methanol and water system. The Rf values in the same solvent system were calculated for V. thapsus fruit (0.26,0.32, 0.46,0.59,0.78) for leaves (0.14,0.22,0.32) for root (0.36,0.46,0.48,0.52, 0.59,0.78) and for stem extract (0.17, 0.39,0.4,0.52, 0.59,0.69,0.78).

Antioxidant activity Antioxidant studies using DPPH, ABTS and Nitric oxide free radical spectrophotometeric methods showed the antioxidant capacity order on % free radical scavenging basis for R. fruticosus various parts extracts as fruit > leaves > root > stem and for V. thapsus root > fruit > leaves > stem. The % radical scavenging capacity using 0.5 mg/ ml of sample in DPPH method were 96%, 92.8%, 90.07% and 89.88% for R. fruticosus fruit, leaves, root and stem respectively. Using same method and concentration gave % radical scavenging for V. thapsus fruit, leaves, root and stem 89.40, 87.73, 96.13 and 85.05 respectively.

Anti-inflammatory activity Formalin induced inflammation in mice and Carrageenan induced inflammation in rats were used to evaluate anti-inflammatory activity. Dose dependent anti-inflammatory effects were observed for both R. fruticosus and V. thapsus at dose of 100, 300 and 500 mg/kg. The fruit and leaves extract of R. fruticosus showed significant anti-inflammatory activity. Leaves extract showed different however the extract of fruit showed the same inhibition in both phases of Formalin test. The root and stem extracts showed no significant anti-inflammatory effect. The reduction capacity of paw edema by R. fruticosus various extracts followed the order; leaves > fruit > root > stem.

The order of potency in formalin test and on the basis of reducing the rat paw edema for V. thapsus various parts using 100, 300 and 500 mg/kg doses were observed as fruit > leaves > root > stem.

Analgesic activity Leaves and fruit exhibited higher of analgesic effect compared to root and stem extracts of R. fruticosus using hot plate, tail flick and writhing test. The order of potency for analgesic effect for various parts of V. thapsus was observed as leaves > fruit > root > stem. The analgesic effect was observed in dose dependent fashion and the applied doses were 100, 300 and 500 mg/kg.

Diuretic activity Albino mice were used to monitor the diuretic activity of R. fruticosus and V. thapsus . Fruit and to some extent leaves of R. fruticosus showed diuretic action but the rest parts showed no significant action. Similarly fruit of V. thapsus was observed for significant diuretic effect but leaves, root and stem showed no significant diuretic activity.

Antibacterial activity and Antifungal activity E. coli , S. typhi, S. aureus, M. luteus, Citrobacter, B. subtilis, P. aeruginosa and P. mirabilis were use in antibacterial studies and significant results were obtained for extracts of both plants using agar disc diffusion method. The order of activity for R.

x fruticosus was stem > leaves > fruit > root and for V. thapsus was leaves > fruit > root > stem. The stem extract of R. fruticosus was found the most effective with MIC of 20µg comparable with standard. No significant antifungal activity was observed for methanolic extracts of both plants.

Gross behavioral studies At dose of 100, 300 and 500 mg/kg the gross behaviors such as sweating, salavation, piloerection and increased respiration etc. were observed. Slight urination was found in fruit of the R. fruticosus . A slight increase in overall performance was observed. Plioerection and cough reflex was observed for leaves extract of V. thapsus and overall decrease in performance was observed.

Neuropharmacological activity Slight anxiolytic effect without sedation was observed with the administration of extract of R. fruticosus and no muscle relaxing effect. Antidepressant effect was observed after forced swimming test. The order of antidepressant effect for various parts of R. fruticosus were as fruit > root > leaves > stem. Sedative effect was observed with V. thapsus especially with fruit and leaves extracts which resulted decrease in motor performance. There was no muscle relaxing effect. Similarly antidepressant effects were produced by leaves and fruit in forced swimming test comparative to root and stem.

Toxicity studies Various parts extracts of R. fruticosus and V. thapsus showed no significant insecticidal activity however the anthelmintic activity was remarkable especially for fruit and leaves of V. thapsus at dose of 50, 75 and 100mg. The brine shrimp toxicity test also point out leaves and fruit of V. thapsus the most cytotoxic. The R. fruticosus was comparatively safer.

INTRODUCTION

Plants have been utilized as medicines from the very beginning of human civilization (Samuelsson, 2004, Balunas and Kinghorn, 2005). The use of plants is simply in decoction, poultice, powder or other crude form to improve poor health conditions (Balick and Cox, 1997; Samuelsson, 2004; Balunas and Kinghorn, 2005). However in recent times the approach is changing with the advancement in isolation and analytical technology but still it is based on old traditional use of medicinal plants (Balunas and Kinghorn, 2005) because almost 80% of the world population depends on traditional system of health care (Ahmad, 1999; Shah and Khan, 2006).

There are different approaches that how these plants are selected as a candidate in drug discovery; these approaches includes random selection for phytochemical screening or random selection followed by biologic assay, the most common approach that mostly followed is based on traditional medicine (ethanomedical) (Fabricant and Farnsworth, 2001). There is increase in the use of traditional medicine throughout the world so its efficacy, safety and quality control is of prime importance (Anderson et al ., 2000).

Presently the quality control checking of traditional medicine is based on conventional marker approach that has the advantage of rapidity and simplicity but there is no universality of this approach because plants contains dozen of chemical constituents with different pharmacological activity (Mok and Chau, 2005). However various traditional monographs recommend the use of this approach for standardization (Thai herbal pharmacopeia, 2000; Beck, 2001). If it is not possible to isolate or identify an active component of an herbal drug then World Health Organization and European medicine agency also suggest for the use of chromatographic finger printing as a quality criteria tool.

Pakistan is so rich in medicinal plants that only the northern areas contain 25000 species out of which 10,000 are important both medicinally and economically (Pei,

1992; Qureshi et al ., 2006). These medicinal plants are still play important role in the treatment of various disorders in rural areas of Pakistan (Qureshi and Ghufran, 2005). But mostly research activities regarding medicinal plants in Pakistan are only limited to the documentation of their traditional knowledge (Shinwari, 2010) and the exploited species are very few.

So we have selected two species Rubus fruticosus and Verbascum thapsus from northern area District Dir of KPK Pakistan, not only to document their traditional uses but to exploit their various parts for various pharmacological activities, toxicities and to evaluate their medicinal uses.

Rubus fruticosus L. Kingdom: Plantae Division: Magnoliophyta Class: Magnoliopsida Order: Rosales Family: Rosaceae Genus: Rubus Species: fruticosus Botanical name: Rubus fruticosus L.

R. fruticosus leaves and stem R. fruticosus fruit and leaves

Common names: Blackberry, bramberry, brambleberry, brummel bramble (Hummer and Janick, 2007) shrubby blackberry and wild blackberry complex. Local names: Karwara (Zabihullah et al ., 2006; Murad et al ., 2011), Ach (Sher and Hussain, 2009) Akhara (Ajaib et al., 2010) and Baganrra (Sher et al ., 2010; Murad et al ., 2011).

Etymology

The word Rubus is a Latin word meaning bramble and fruticosus means bushy.

Part Used: Leaves, fruit and root

Distribution R. fruticosus grows wild in Northern areas of Pakistan like Chitral (Ahmad et al ., 2006), Dir (Jan et al ., 2008), Mansehra (Shah and Khan, 2006), Malakand (Zabihullah et al. , 2006; Sher et al ., 2011) and Kotli (Ajaib et al ., 2010). However it has a center of origin in Armenia. It is well distributed throughout Europe and Morocco. It has been introduced into Asia, Oceania and North and South America (Swanston- Flatt et al ., 1990; Pullaiah, 2003; Hummer and Janick, 2007).

History Ancient cultures explored R. fruticosus as wild plant. The Greeks used it to treat gout. Romans treated various diseases through the use of tea prepared from blackberry leaves (Malcolm, undated). R. fruticosus as food was used about 8,000 BCE and as medicinal plant for native peoples soon after the Ice Age (Connolly, 1999). Hippocrates used blackberry leaves and stems soaked in white wine, as an astringent poultice on wounds and in difficulties in childbirth (Littre, 1979). Ancient Egyptians were aware about the use of blackberry but they did not document their uses. Egyptian used “aimoios ” or “ametro s” terms for blackberry about the second century CE (Manniche, 1989; Hummer and Janick, 2007).

Botanical Description R. fruticosus is a bushy plant having thorns but some cultivated varieties are free of thorns. Blackberries are perennial lasting three seasons or more (Hummer and Janick, 2007).

Flowers The flowers are produced in late spring and early summer. Each flower is about 2- 3 cm in diameter with five white or pale pink petals. Flowers have five petals, multiple stamens and are usually white though sometimes pink. As the petals fall, the fruit develops an aggregate of drupelets that are green in beginning and red to black when ripens. Flowers and fruit occur in a panicle-like or racemose-cymb (Hummer and Janick, 2007).

Fruit Generally the fruit is termed as berry or blackberry. It is a dense cluster of separate units or drupelets to form fruit which on ripening turn black or dark purple from red (Hummer and Janick, 2007).

Leaves Leaves are dark green in colour on top with a lighter green underside. The veins and stalks of leaves are covered with short prickles. Leaves tend to be ternate above, tending to five palmate leaflets or sometimes seven towards the base. Adaxial sides of leaflets are fold into pleats and glabrate which are green in summer, darkening red-purple in the fall, and deciduous in winter (Hummer and Janick, 2007).

Stem Plant typically bears biennial stems or semi woody called canes. They vary from sprawling to almost erect, spreading shrubs with thorn and leaves, the stem grow up to 7 m in length that is greenish, purplish or red in colour. Young canes emerge from buds on the woody root each spring and grow very rapidly i.e. 50 –80 mm a day (Hummer and Janick, 2007).

Chemical Constituents The important chemicals that are isolated are triterpenes, sterols (Liu et al ., 1993; Mingsheng, 1994; Durham et al ., 1996; Shepherd et al ., 1999), triterpene acid (Mukherjee et al ., 1984; Mingsheng, 1994), glycosides (Haddock et al ., 1982; Gupta et al ., 1982) and anthocyanins (Robinson and Robinson, 1932; Markides, 1982; Goiffon et al ., 1991 ; Dugo et al ., 2001; Stintzing et al ., 2002; Talaveara et al ., 2005; Wu and Prior, 2005; Fan-Chiang and Wrolstad, 2005; Elisia et al ., 2007, Elisia and Kitts, 2008; Ogawa et al ., 2008). Cyanidin-3-glucoside is the most dominant one i.e. 87.5% of total anthocyanins (Wu and Prior, 2005). The fruit of blackberry is quite rich in vitamin C (Benvenuti et al ., 2004; Szajdek et al ., 2008). Apart from the above explored constituents the plant also contain

5 phyto-oestrogen (Mazur et al ., 2000), fatty acid (Hoed et al ., 2009), sterols, tocols (Adhikari et al ., 2008, Hoed et al., 2009), flavonoids (Gudej and Tomczyk, 2004), carotenoids (Marinova and Ribarova, 2007) and rare earth elements (Wyttenbach et al ., 1998).

Medicinal Uses

The root-bark and the leaves are strongly astringent, depurative, diuretic, tonic and vulnerary (Launert, 1981; Grieve, 1984; Chiej, 1984; Mills, 1988; Chevallier, 1996). It is used as excellent remedy for dysentery, diarrhoea, haemorrhoids, cystitis etc. The root is more astringent (Grieve, 1984; Bown, 1995; Murad et al ., 2011). Externally it is used as a gargle to treat sore throats, mouth ulcers and gum inflammations (Bown, 1995; Chevallier, 1996). A decoction of the leaves is useful as a gargle in treating thrush and also makes a good general mouthwash (Chiej, 1984). The fruit juice is used to treat asthma (Murad et al ., 2011). The leaves of the plant are also used in various respiratory problems (Blumenthal, 1998).

General Uses

Fruit are eaten raw or cooked (Loewenfeld and Back, 1974; Mabey, 1974; Chiej, 1984; Launert, 1981; Facciola, 1990). Syrups, jams and other preserves are prepared from fruit of R. fruticosus (Bown, 1995). The cooked root are also used as food (Chiej, 1984) while leaves whither dried or fresh are used as tea (Usher, 1974; Lust, 1983). The young shoots are peeled and consumed in salads (Phillips and Foy, 1990).

Verbascum Thapsus L. Kingdom: Plantae Division: Magnoliophyta Class: Magnoliopsida Order: Scrophulariales Family: Scrophulariaceae Genus: Verbascum Species: thapsus Botanical name: Verbascum thapsus L.

Flower of V. thapsus Leaves flower and stem of V. thapsus

Common names : Adam's flannel, Aaron's rod, Beggar's blanket, Beggar's stalk, Big taper, Blanket herb, Bullock's lungwort, Candlewick plant, Clot, Clown's lungwort, Common mullein, Cuddy's lungs, Duffle, Feltwort, Flannel mullein, Flannel plant, Flannel leaf, Fluffweed, Golden rod, Hare's beard, Hag's taper, Jacob's staff, Jupiter's staff, White mullein, Mullein dock, Old man's flannel, Our lady's flannel, Peter's staff, Rag paper, Shepherd's staff, Shepherd's clubs, Torches, Velvet dock, Velvet plant, Woollen and Wild ice leaf. So many names indicate friendly, majestic, plain old and soft image of the plant in minds

(Britton and Brown, 1898; Durant, 1976; Brickell and Zuk, 1997; Wagner et al ., 1999).

Local names : Kharghwug (Murad et al ., 2011), Ghordoughkaro (Hussain et al ., 2007), Gidder Tambakoo (Qureshi et al ., 2007), Tamakusak (Shinwari and Gilani, 2003), Khardhag (Sher, 2011), Jungle tambako and barbasco (Pullaiah, 2003).

Parts Used: Leaves, flowers and roots

Etymology

The Verbascum is derived from Latin word barbascum (Latin barba or beard) (Jankowiak, 1976) referring to the plant's beardlike filaments (Wilhelm, 1974). Thapsus is considered to be derived from the Siclian Isle of Thapsos where mullein was gathered in ancient times (Muenscher, 1935) or from the Tunisian Island “Thapsus ” (Jankowiak, 1976). “Thapsos ” is Greek word representing the yellow flower of the plant (Wilhelm, 1974). The plant has yellow flowers so Roman ladies dyed their hair yellow (DeBray, 1978). The word mullein is derived from the Latin mollis, or soft, (Durant, 1976) which is synonymous to the present term woolen (Muenscher, 1935; Mitich, 1989).

Distribution Verbascum thapsus is distributed in different Pakistani areas like Kurram Agency, Dir, Chitral, Swat, Gilgit, Deosai, Baltistan, Drass, Ladakh Hazara, Poonch, Kashmir, Baluchistan and Punjab (Shinwari and Gilani, 2003). It is widely distributed plant, being found all over Europe and in temperate Asia as far as the Himalayas, abundant in North America as a naturalized weed in the eastern States (Hoshovsky, 1986; Murbeck, 1933; Ansari and Daehler, 2000).

History Common mullein has an old relationship with man. It has respected because of mystical and medicinal powers traditionally. According to old herbalists if some one keeps a part of the plant it has the ability to keep away evil spirits and terrors (Muenscher, 1935; DeBray, 1978). It was famous among Greeks that Ulysses took this plant to protect himself against the wiles of Circe (DeBray, 1978). Greeks, Romans and people of Western United States knew it as candle/torch so they used it at funerals or other holy ceremonies (Muenscher, 1935; Mitich, 1989). The people of Rome and Ireland called it “lungwort” because of its use to cure lung disease in both humans and livestock (DeBray, 1978; Muenscher, 1935). Few folk`s names of the plant shows its association with Christianity and medieval beliefs in witches those are Lady`s candle, Lady`s flannel, Our Lady`s taper, Lady`s candlestick, Virgin Mary`s candle, Wooly mullein and Hag`s Taper (McLeod, 2008).

Botanical Description V. thapsus is an herbaceous biennial or annual, erect and stout weed. It produces a low vegetative rosette up to 24 inches.

Flower Flowers are densely arranged, occur usually one per 5 axil having both male and female reproductive organs. Flowers are yellow in colour having five sepals, five petals, two-celled ovary and five stamens.

Fruit Fruit is in a form of capsule that split into two valves at maturity. Capsule has star shaped appearance and ovoid having 3-6 mm length. The fruit contain seeds that are brown in colour having 0.5-1.0 mm length.

Leaves Basal leaves are oblong-obovate to oblanceolate generally entire, having short and long petioles (10-40 cm). Leaves margins are entire or obscurely crenate and alternately arranged. Leaves arranged along the stem (cauline leaves) are 2-12 inches in length having pinnate venation.

Roots Mullein has a deep taproot with a shallow secondary fibrous root system.

Stem Stem is erect and stout having 50 to 180 cm range of tallness. The stem is generally simple having alternately arranged leaves (Remaley, 1998; Wagner et al ., 1999; Halvorson and Guertin, 2003).

Chemical Constituents The mullein plant contains various chemical constituents like saponins e.g. triterpene B, triterpene A, saikogenin A (Pascual Teresa et al ., 1978); thapsuine B, hydroxythapsuine, thapsuine A, hydroxythapsuine A (Pascual Teresa et al ., 1980); iridoid glycosides e.g. aucubin and isocatalpol and their various deravitives (Seifert et al ., 1985), verbascoside A and phenylethanoid glycosides (Warashina et al ., 1991, 1992), harpagide (Bianco et al ., 1984), flavonoids (Souleles and Geronikaki, 1989), vitamin C (Kellermann, 1944; Franjo, 1950) and elements (Petrichenko and Yagontseva, 2006).

Medicinal Uses The flowers and leaves are analgesic, anti-inflammatory, antiseptic, spasmolytic, astringent, diuretic, emollient, expectorant and vulnerary (Uphof, 1959; Triska, 1975; DeBray, 1978; Lust, 1983; Chiej, 1984; Grieve, 1984; Mills, 1985; Foster and Duke, 1990; Hussain et al ., 2007). Homeopathic formulations containing fresh leaves are used in the treatment of long-standing headaches accompanied with oppression of the ear (Grieve 1984). Ointments prepared from leaves are

10 used for burns and earache (Haughton, 1978). Topically, a poultice of the leaves is a good healer of wounds and is also applied to ulcers, tumours and piles. A poultice made from the seeds and leaves is used to draw out splinters (Grieve, 1984).

Infusion of the flowers in olive oil is used as earache drops having strong bactericidal properties (Grieve, 1984; Foster and Duke, 1990; Bown, 1995; Chevallier 1996). An infusion or tea of the plant is taken internally in the treatment of a wide range of chest and abdominal complaints including productive cough and diarrhea (DeBray, 1978; Haughton, 1978; Grieve, 1984; Bown, 1995; Chevallier, 1996; Murad et al ., 2011).

It is used as a tobacco substitute (Wilhelm, 1974) and rheumatic problems (Haughton, 1978). A decoction of the seeds is used to soothe chilblains and chapped skin (Chiej, 1984).

The juice of the plant and powder made from the dried roots is said to quickly remove rough warts when rubbed on them (Grieve, 1984). A decoction of the roots is said to alleviate toothache and also relieve cramps and convulsions (Grieve 1984).

General Uses Flower extract in hot water is used as dye to turn hair a golden colour (Grieve, 1984; Huxley, 1992) that can be changed to green dye on acidification or to brown on alkalization (Polunin, 1969; Grae, 1974; Triska, 1975; Grieve, 1984).

Leaves are used as insulation in shoes to keep the feet warm (Grieve, 1984; Huxley, 1992). Mullein was also used as a useful fish poison (Haughton, 1978).

The stems are used as torches, tinder and wicks for candles (Johnson, 1862; Chittendon, 1956; Polunin, 1969; Coon, 1975; Triska, 1975; DeBray, 1978; Grieve, 1984; RHS, 1988).

LITERATURE SURVEY OF RUBUS FRUTICOSUS L.

Pharmacological literature survey of R. fruticosus

Activity or Model of study Results Reference Part used

Antimicrobial activity Juice B. subtilis, B. cereus, E. Inhibition (Krisch et al ., 2008) coli and S. marcescens between 50% and 75% Fruit cordials Bacteria Active partially (Cavanagh et al., 2003)

Fruit extract P. aeruginosa 10116-195 Antiviral active (Corao et al ., 2002) fractions bacteriophage

Methanolic M. tuberculosis MIC of 1mg/ml (Rios et al ., 1987) extract of in agar dilution aerial parts test Antioxidant activity Ethanolic DPPH free radical method used in beverages (Buřičova and extract of and other Reblova, 2008) leaves nutrients Fruit Rats Chemopreventive (Stoner et al ., 2006) Fruit ORAC method 14.8 –22.6 μmol (Jiao and Wang, Trolox/g fresh 2000) weight Anti-inflammatory activity Fruit Murine model (in vivo) Anthocyanin as (Dembinska-Kiec et active al., 2008) Fruit aqueous Hyaluronidase enzyme (in Active (Marquina et al.,

13 extract vitro) 2002) GOD type Hyaluronidase enzyme Active (Nakahara et al ., tannin inhibition 1998) Antidiabetic activity Fruit (aqueous No anti-diabetic effect in Single (Büyükbalci and tea) vitro glucose diffusion Sedef , 2008) model Aqueous and Noninsulin dependent Single (Xu et al ., 2006) butanol diabetes extracts of leaves Aqueous Rats Active (Jouad et al ., 2002) extract of leaves Tea made Chromium and zinc Single (Castro, 2001) from leaves dependent diabetes Fruit Mice No affect on (Swanston-Flatt et glucose al ., 1990) homeostasis Leaves Alloxan-diabetic rabbits Decrease glucose- (Alonso et al., 1980) induced hyperglycemia Leaves Alloxan- diabetic rats Effective (Sharaf et al ., 1963) antidiabetic

Therapeutic application (Single or in combination with other plants)

Plant part Main therapeutic Single or in Reference ailment com bination / patent Leaves Deodorant Part of patent (Takase et al ., 2011) Extract of Skin aging Part of patent (Kizoulis et al ., 2010) leaves Leaves Diarrhoea, dysentery Single (Tiwari, 2008) Twig tops Diarrhea, menstruations Single (Leonti et al., 2009) (decoction) Chewing the strengthens the gums and Single leaves cures thrush (Leonti et al ., 2009)

Leaves as abscesses and fungal Single (Leonti et al., 2009) wrapper infection Health Vitamins and mineral Combination (Zhou et al ., 2008) granules supplement, immunity /patent enhancer formulation Leaves extract Anti-aging Single (Herrmann, et al ., 2007) A part of Cardiovascular In combination (Randolph and Roh- herbal product conditions, arthritis, with other Schmidt , 2007) osteoporosis and plants Alzheimer's disease Leaves extract Oral and pharyngeal Single/Patent (Vielhaber et al., 2007) cavity treatment and to slow down aging Aqueous Angiogenic property Gallic acid as (Greenway et al ., 2007) extract of active leaves Whole plant Dental carries, gums and Toothpaste (Abbruzzo and Ignazio,

cleaning teeth formulation in 2006) combination Powdered Nutritional supplement In combination (Liu et al ., 2005) fruit Extract of Anti-influenza In combination (Shimizu et al ., 2004) whole plant Extract whole Prevention and treatment Patent (Koch et al., 2004) plant of inflammatory, immune formulation in and metabolic diseases combination Fruit Antidiarrhoeic, treat Single (Guarrera, 2003; Pieroni throa t inflammation, et al ., 2004) astringent, diuretic , anti- bruises and anti- haemorrhoids Extract of the Deodorant against allyl In combination (Shimizu et al ., 2003) whole plant methyl monosulfide Powdered Digestive disorders in In combination (Grigore et al ., 1981) leaves calves and piglets Whole plant Diuretic and A part of (Ninova et al., 1980) hypoazotemic activities Nephroton (product )

Literature data for total polyphenols, total anthocyanins and ascorbic acid

Number Total Total Ascorbic Reference of polyphenols anthocyanins acid cultivars (mg/100 g) (mg/100 g) (mg/100 g)

4 2030 134- 152 15.22 (Pantelidis et al ., 2007)

7 289.3 88.7 12.9 (Benvenuti et al ., 2004)

27 460 141 NR (Moyer et al ., 2002)

2 417 –555 110-122 NR (Sellappan et al ., 2002)

6 NR NR 14.9 (Jiao and Wang, 2000)

3 226 152.8 NR (Wang and Lin, 2000)

1(Chester) 361 NR NR (Heinonen et al ., 1998)

5 320 80 20.4 (Rotundo et al ., 1998)

4 07.5 115 NR (Costantino et al ., 1992)

2 NR NR 6 (Romero Rodriguez et al ., 1992) NR stands for not reported

Phytochemical literature survey of Rubus fruticosus

Compounds Plant parts/Group Reference

Organic acids Citric acid Fruit (Denev et al ., 2010) Malic acid (Kafkas et al ., 2006) Galacturonic acid (Haminiuk et al ., 2006) Organic acids Fruit powder (Liu et al ., 2005)

Carbohydrates Glucose Fruit (Denev et al ., 2010) Fructose (Kafkas et al ., 2006) Sucrose (Milivojevic et al ., 2011) Galactoglucomannan Suspension culture of (Cartier et al ., 1988) cell wall Pectins Fruit (Haminiuk et al ., 2006) Vitamins α-tocopherol Seed oil, seeds from (Adhikari et al ., 2008; Hoed γ-tocopherol Korean Thornless et al ., 2009) γ-tocotrienol blackberry δ-tocopherol (Liu et al ., 2005) Vitamins Fruit powder (Stoner et al ., 2006) A Fruit C E Folic acid Steroids Desmethylsterols Seed oil (Hoed et al ., 2009) Campesterol Stigmasterol ( Δ5) β-sitosterol ( Δ5)

Δ5-avenasterol Δ7-sitosterol Δ7-avenasterol Fruit (Mazur et al ., 2000) Squalene Daidzein Geniste in Secoisolariciresinol Matairesinol Lipids Lauric acid Seed oil (Hoed et al ., 2009) Myristic acid Palmitic acid Stearic acid Oleic acid Linoleic acid α - linolenic acid Arachidic acid Saturated fatty acids Monounsaturated fatty acids Leaves (Haas and Rentschler, 1984) Polyunsaturated fatty acids Wax. (Free alcohols, Fatty acids, esters, alcoholic acetates Anthocyanin Cyanidin-3-glucoside Fruit (Fan-Chiang and Wrolstad, 2005; Talavera et al ., 2005; Wu and Prior, 2005; Elisia and David, 2008; Ogawa et al ., 2008) Cyanidin-3-dioxaloylglucoside Fruit (Fan-Chiang and Wrolstad, 2005; Talavera et al ., 2005; Wu and Prior, 2005)

Cyanidin-3-xyloside Fruit (Fan-Chiang and Wrolstad, 20 05; Talavera et al ., 2005; Wu and Prior, 2005 ; Ogawa et al ., 2008; Elisia and David , 2008) Cyanidin-3-arabinoside Fruit (Ogawa et al ., 2008) Cyanidin-3-rutinoside

Cyanidin-3-(6-malonoyl) glucoside Fruit (Elisia and David, 2008) Pelargonidin-3-O-glucoside Malvidin-3-O-glucoside

Cyanidin-3-pentose Fruit (Talavera et al ., 2005) Cyanidin -3-malonylglucoside

Cyanidin-3-xyloside Fruit (Wu and Prior, 2005) Cyanidin -3-(6-malonyl) glucoside Cyanidin-3-pentose Fruit (Talavera et al ., 2005) Cyanidin-3-malonylglucoside Cyanidin-3-rutinoside Fruit (Markides, 1982; Dugo et al ., Cyanidin-3-xyloside 2001; Stintzing et al ., 2 002; Malvidin-3-glucoside Fan -Chiang and Wrolstad, Cyanidin 3-malonylglucoside 2005) Cyanidin-3-dioxalylglucoside Cyanidin 3-saccharide Stems and (Robinson and Robinson, leaves 1932) Carotenoids Lutein Fruit (Marinova and Ribarova, Zeaxanthin 2007) Lycopene

β-cryptoxanthin α-carotene β-carotene

Elements Lead (Pb) Shoots, roots (Yoon et al ., 2006) La, Ce, Nd, Sm, Eu, Tb, Yb, Lu Leaves (Rare (Wyttenbach et al ., 1998; earth elements) Castro, 2001) Chromium Leaves (Castro, 2001, Toth et al ., 2008) Zinc Fruit, leaves (Toth et al ., 2008) Manganese Ca lcium Copper Tin Iron Nickle Flavonoids Quercetin Leaves (Gudej and Tomczyk, 2004; Hyperoside Sanjust et al ., 2008) Kaempferol Fruit Morin Myricitin (Milivojevic et al ., 2011) Tanninns Ellagic acid Fruit (Lei et al ., 2001; Carlsen et al ., 2003) GOD type ellagitannin Leaves (Nakahara et al ., 1996) Gallic acid Leaves/fruit (Lei et al ., 2001;Greenway et al ., 2007) Aromatic compounds Furans Fruit (Turemis et al ., 2003) 5-hydroxymethylfurfural 2, 3 -dihydro-3, 5- dihydroxy-6-methyl- 4H -pyran-4-one

Triterpene acid Rubutic acid Leaves (Mingsheng, 1994) Rubinic acid Leaves (Mingsheng,1994; Mukherjee et al ., 1984) β-Amyrin (Shepherd et al ., 1999) 2-α-Hydroxyursolic acid (Mingsheng, 1994 )

Glycosides β-1-O-Galloyl-2,3:4,6-bis - Leaves (Gupta et al ., 1982) hexahydroxydiphenoyl-D-glucopyranose α-1-O-Galloyl-2,3:4,6-bis - hexahydroxydiphenoyl-D-glucopyranose β-1,2,6-Tri-O-galloyl-D-glucose (Haddock et al ., 1982) β-Penta-O-galloyl-D-glucose Quercetin 3-β- Glucuronides Leaves and fruits (Henning, 1981) Quercetin 3-β-glycosides Quercetin 3-xylosylglucuronides Kaempferol-3-β- Glucuronides Kaempferol 3-β-glycosides Kaempferol 3-xylosylglucuronides

Structures of different chemical constituents reported from Rubus fruticosus

LITERATURE SURVEY OF VERBASCUM THAPSUS L.

Ethnobotanical literature survey

Part used Medicinal use in different location Reference Leaves, flower Skin diseases, cuts, wounds and (Sher, 2011) swelling (in Malakand, Pakistan) Whole plant Ear problems of cats and dongs in (Lans et al ., 2008) Canada Whole plant Inflammatory ailments in respiratory (Rodriguez- tract and others (in Mexico) Fragoso et al ., 2008) Leaves Antitussive by Albanian and Italian (Pieroni and Quave , 2005) Infusion from In combination for dental treatment in (Anderson, 2004) root Medieval England Leaves Ear disorders (Nakar, 2004) Flower and leaves Pulmonary disease, fever and bleeding (Shinwari and Gilani, of lungs and bowel in Pakistan 2003) Whole plant Naturopathic herbal extract ear drops (Michael et al ., 2003) as immunity enhancer Ointment, Antitussive in Bulgaria (Leporatti and decoction in olive Ivancheva , 2003) oil Flower Emollient against catarrh and coughs. (Leporatti and Cataplasm applied externally to soothe Ivancheva , 2003) gangrenous sores in Italy Leaves Tobacco substitute (Coy-Herbert, 2002) As part of Skin moisturizing and conditioning (Ohara et al., 2001) polyherbal effect s formulation

Leaves Diarrhea and dysentery in cattle (Shinwari and Khan, 2000) Islamabad, Pakistan Alcoholic extracts Inhibit viral infectivity (Zanon et al ., 1999) Oil from flower For treatment of piles, bruises and (Grieve, 1995) frostbites in Germany Leaves Ointments for burns and earache in (Haughton, 1978) Eastern United States. Tea For lung disease in ancient Rome and (DeBray, 1978) now in modern Ireland Dried flower or As cigarettes for asthmatics by Indians (Lewis and Elvin- root and Menominee (Michigan) Lewis, 1977) Tincture from Promote healthy hair in baldness and (Roia, 1966) dried leaves dandruff preparations (US) Mullein-tops with Asthma and hemorrhages (Nichols, 1887) other plants As a part of All bodily disorders strains, bruises, (Baker, 1885) healing plaster fresh wounds, inflammation, rheumatic, neuralgic, and all chronic pains, ulcerations Iodine and In combination for throat disease (Humphrey, 1869) mullein

Pharmacological literature survey

Activity/Part Model of study Results Reference used Stem extract B. subtilis, Antibacterial (Kumar and Singh, (Alcoholic) P. aeruginosa, S. 2011) aureus, E. coli Stem extract DPPH assay Antioxidant (Kumar and Singh, (Alcoholic) 2011) Ethanolic DPPH assay Up to 85% (Narayanaswamy and extract inhibition of free Balakrishnan, 2011) radical Water extract Up to 40% inhibition Mullein herb Clinical trials Anti-tubercular (Quinlan, 1883; Carthy positive results and Mahony, 2011) Mullein flower Wound model in Wound healing (Mehdinezhad et al ., rabbits stimulatory effect 2011) Aqueous extract In vitro enzyme Anticancer activity (Cyr, 2010) and alcoholic inhibition assay: extract (MMP-9 ) and Cathepsin B Aerial parts Dye uptake Antiviral active (Rajbhandari et al ., assay against influenza 2009) virus A A part of hair 5-alpha.- Hair outer root (Ito et al ., 2007) tonic (Leaves) reductase sheath proliferation inhibitors stimulators, and hair tonics Aqueous and K. pneumoniae , Antibacterial (Turker and Camper, alcoholic E. coli , P. 2002)

30 extract aeruginosa , S. aureus etc. Aqueous and Potato disc Tuomour inhibitory (Turker and Camper, alcoholic method 2002) extract Aqueous and In vitro Inhibit seed (Turker and Camper, alcoholic germ ination in 2002) extract large doses Boiling water Human liver Anti-hepatoma (Lin et al ., 2002) extract of aerial cancer cell lines active in vitro parts HA22T/VGH, growth inhibition Hep3B and From 31.0 to PLC/PRF/5 69.9%. (anti-hepatitis B) Polysaccharides Rats Decrease in (Aboutabl et al., 1999) from leaves cholesterol and triglyceride levels Aqueous and Antioxidants Inhibit sebum (Ota et al ., 1999) ethanolic oxidation thus extract of whole preventing body plant smell and skin aging Methanolic Herpesvirus type Active (McCutcheon et al., extract of whole 1 1995) plant Decoction of Virus Antiviral active (Skwarek, 1979) flowers

Phytochemical literature survey of Verbascum thapsus

Compounds Plant Reference parts/Group Carbohydrates Verbascose Roots (Murakami, 1940) Stachyose Roots (Hattori et al., 1958) Heptaose Octaose Nonaose Sugars Trichomic cells (Miroslavov and Komarov, 1959) Sucrose Seed oil ( Pascual Teresa et al ., 1978) Pectins Roots ( Verdon, 1912)

Elements and radicals Basic elements Ash of fruit ( Pande and Tewari, 1960) Al Na K Mg Acidic radicals carbonate sulfate chloride silicate Cr and Ni Whole plant (Kfayatullah et al ., 2001) Zn and Pb Whole plant (Shah et al., 2004) Lipids Linoleic acid Seed oil (Petrichenko and Razumovskaya, 2006 )

Oleic acid Fruit, seed oil (Pande and Tewari, 1960; Stearic acid Pascual Teresa et al ., 1978) Palmitic acid β -sitosterol Arachidic acid Seed oil (Pascual Teresa et al ., 1978) Behenic acids Ergosta-7-en-3 -β-ol Saponins Triterpene B Aerial part (Pascual Teresa et al. , Triterpene A 1978) Saikogenin A Veratric acid Benzene extract (Pascual Teresa et al ., 1978) β-spinasterol 6-O-β-D-xylopyranosyl aucubin Aerial parts (Bianco et al ., 1980) Thapsuine B Aerial parts (Pascual Teresa et al ., 1980) Hydroxythapsuine Thapsuine A Hydroxythapsuine A Aerial parts (Ding et al ., 1986; Zhao et 3-O-fucopyranosylsaikogenin F al. , 2011) Iridoid glycosides Harpagide Aerial parts (Bianco et al ., 1984) Aucubin Roots (Groeger and Simchen, 1967; Seifert et al ., 1985; Pardo et al ., 1998) Isocatalpol Whole plant (Seifert et al ., 1985) Isocatalpol Methylcatalpol 6-O-α-L-rhamnopyranosylcatalpol Laterioside Roots Pardo et al ., 1998) Ajugol Roots, A erial Pardo et al ., 1998; Zhao et

parts al ., 2011)

Picroside IV Whole (Tatli et al ., plant 2004; Hussain

et al ., 2009) Saccatoside (Warashina et 6-O-(3” -O-p-coumaroyl)-α-L-rhamnopyranosylcatalpol al ., 1991) 6-O-(4” -O-p-coumaroyl)-α-L-rhamnopyranosylcatalpol

6-O-(2” -O-(p-methoxy-trans-cinnamoyl)-α-L- rhamnopyranosylcatalpol 6-O-(3” -O-(p-methoxy-trans-cinnamoyl)-α-L- rhamnopyranosylcatalpol

Verbascoside Leaves (Murakami and Susumu,

1940)

Verbascoside A (Warashina et Whole 6-O-[2” -O-(3,4-dihydroxy-trans-cinnamoyl)]-α-L- al ., 1991) plant rhamnopyranosylcatalpol 6-O-[4” -O-(3,4-dihydroxy-trans-cinnamoyl)]-α-L- rhamnopyranosylcatalpol 6-O-[3” -O-(3,4-dimethoxy-trans-cinnamoyl)]-α-L- rhamnopyranosylcatalpol 6-O-(2” -O-feruloyl)-α-L-rhamnopyranosylcatalpol 6-O-(4” -O-feruloyl)-α-L-rhamnopyranosylcatalpol 6-O-(2” -O-isoferuloyl)-α-L-rhamnopyranosylcatalpol 6-O-(3” -O-isoferuloyl)-α-L-rhamnopyranosylcatalpol 6-O-(4” -O-isoferuloyl)-α-L-rhamnopyranosylcatalpol

5-O-α-L-rhamnopyranosyl (1 α3)-[α- Whole plant (Mehrotra et al ., 1989) D-glucuronopyranosyl (1 α6)]-α-D-

34 glucopyranoside

Ningpogenin Aerial parts (Yue et al., 2001 ; Zhao et al ., 2011) 10-deoxyeucommiol Aerial parts (Gouda et al ., 2003; Zhao et

al ., 2011) Jioglutolide Aerial parts (Morota et al .,1989; Zhao et

al ., 2011) 6-β-Hydroxy-2-oxabicyclo [4.3.0]Δ8 - Aerial parts (Valladares and Rios, 2007 ; 9-nonen-1-one Zhao et al ., 2011) 8-cinnamoylmyoporoside Aerial parts (Qi et al ., 2006; Zhao et al ., 2011) Flavonoids 3α -hydroxydrimmanyl-8-methanoate Leaves (Khuroo et al ., 1988)

Apigenin-4’ -rhamnoside Leaves (Souleles and Geronikaki, 7, 4’ -dihydroxyflavon-4-rhamnoside 1989) Isorhamnetin Verbacoside Aerial parts (Mehrotra et al ., 1989) Apigetrin (Takeda et al ., 1993; Zhao et al ., 2011) 5-hydroxy-6,7-dimethoxyflavone-3- Leaves (Souleles and Geronikaki, ol 1989) 4', 7-dihydroxyflavone-4'-rhamnoside Leaves and (Souleles and Geronikaki, 6-hydroxyluteolin-7-glucoside flowers 1989) 3'-methylquercetin Acacetin-7-O-α-D-glucoside Aerial parts (Danchul et al., 2007) Luteolin Aerial parts (Mehrotra et al ., 1989; Danchul et al., 2007; Zhao et al ., 2011) Cynaroside Aerial parts (Danchul et al., 2007) Kaempferol Quercetin

Rutin Amentoflavone Whole plant (Hussain et al ., 2009) Pulverulentoside I Whole plant (Warashina et 6-O-(2” -O-p-methoxy-trans-cinnamoyl-4” -O-asetyl)-α- al ., 1991) L-rhamnopyranosylcatalpol Ajugol 6-O-benzoyl ajugol 6-O-syringoyl ajugol 6-O-vanilloyl ajugol Harpagoside (Pardo et al ., 1998; Zhao et al ., 2011) Phenylethanoid glycosides Forsythoside B Whole plant (Warashina et Alyssonoside al ., 1992) Arenarioside 1’ -O-β-D-(3,4-dihydroxy-phenyl)-ethyl-α-L- rhamnopyranosyl-(1→3’) -β-D-xylopyranosyl-(1→6’) - 4’ -O-feruloyl-glucopyranoside Leucosceptoside B 1’ -O-β-D-(3-hydroxy-4-methoxy-phenyl)-ethyl-α-L- rhamnopyranosyl-(1→3’) -β-D-xylopyranosyl-(1→6’) - 4’ -Oferuloyl- glucopyranoside Cistanoside B 1’ -O-β-D-(3,4-dihydroxy-phenyl)-ethyl-α-L- rhamnopyranosyl- (1→3’) -3”’ -hydroxy-4”’ -O-β-D- glucopyranosyl- cinnamoyl-(1→6’) -glucopyranoside Ergosterol peroxide Flowers (Zhang et al ., Docosanoic acid 1996) Oleanolic acid β-sitosterol

Terpenes Buddlindeterpene A Whole plant (Hussain et Buddlindeterpene B al ., 2009) Buddlindeterpene C Miscellaneous compounds (+)-genipin Whole plant (Hussain et α-gardiol al ., 2009) β-gardiol Peroxidase Trichomic (Miroslavov Ascorbic acid cells and Komarov, Tannins 1959)

α –galactosidase Roots (Bom et al. , 1998) Rotenone Leaves (Obdulio and Lobete , 1943)

Structures of different chemical constituents reported from Verbascum thapsus

Experimental Materials and Methods The plant materials were collected from District Dir, Khyber Pukhtun Khwa, Pakistan in 2008-09. The plants Rubus fruticosus L. and Verbascum thapsus L. were identified by Prof. Dr. Mansoor Ahmad, Department of Pharmacognosy, Research Institute of Pharmaceutical Sciences, University of Karachi and voucher specimen RIPS-200812, RIPS-200813 were deposited in the laboratory of Research Institute of Pharmaceutical Sciences, University of Karachi, Karachi, Pakistan.

For the preparation of bacterial agar media, Tryptic soya agar (Difco, USA), Bactotryptone (USA), Bactosoytone (USA), sodium chloride (Merck, Germany), Bacto agar (USA) were used.

For the preparation of fungal growth agar media, Sabouraud Dextrose media, mycological peptone (UK), glucose agar (UK) were used.

Mice (25-30g) and rats (200-250 gm) were purchased from animal house of Research Institute of Pharmaceutical Sciences (RIPS).

TLC plates

(1) 20 20 cm, silica gel 60 pre-coated plates, fluorescence at 254 nm (Merck, Germany).

(2) 5 10 cm, silica gel 60 T254 (0.2 mm thick) pre-coated TLC plates, fluorescence at 254 nm (Merck, Germany).

TLC plates were used for detection of chemical constituents.

Drug: Amoxil (amoxicillin) 25 mg, Ampicillin 500 mg injection (both from Smith Kline Beecham Pharmaceutical, Pakistan), Itraconazole and Amphotericin B. It was used as a control in microbial assay. Niclosamide, Caffeine, Diazepam, Codeine, Aspirin.

Solvents: Ethanol, Methanol, Chloroform, Ethylacetate (Merck)

Instrumentation Apparatus: 1. Rotary evaporator, Eyela (Japan) was used for extraction process for dying the extract under reduced pressure. 2. Sonicator (UK) was used for mixing or dissolving the extracts and compounds. 3. Autoclave (Pakistan) was used in microbial assay for sterilization of materials or apparatus. 4. Incubator (Sieco, China) was also used in microbial assay for incubation of microbes. 5. Shaker SS-80 (Japan) was used for shaking the mixtures of solvents and plants extracts. 6. Hotbox oven (Gallenkamp, UK, size two) was used for sterilization of materials or apparatus.

7. Micro Pipette Justor ™, Japan was used in toxicity test for LD 50 . 8. U.V. lamp 1. U.V. lamp: (original Hanau 254 nm, Fluotest) 2. U.V. lamp: Lamp s/w = G8T5 Lamp L/w = TL8w/08 (London).

U.V. lamps were used for detection of compound spots present on TLC plates. 9. Physical balance 1. Libror AEG-120, Shimadzu (Japan). 2. Libror EB-3200 D, Shimadzu (Japan).

Physical balances were used for weighing during experiments. 10. Filter units, Sartorius (Minisart NML, SM 16534, USA): Filter units were used for filtration of extract solution during microbial assay. 11. Head dip box, open field, home cage, traction table, water bath, analgesiometer, swimming tank, stop watch, Dissection box.

Abbreviations used RFF Fruit (extract) of Rubus fruticosus RFL Leaves (extract) of R. fruticosus RFR Root (extract) of R. fruticosus RFS Stem (extract) of R. fruticosus VTF Fruit (extract) of Verbascum thapsus VTL Leaves (extract) of V. thapsus VTR Root (extract) of V. thapsus VTS Stem (extract) of V. thapsus HCl Hydrochloric acid HOAc Acetic anhydride MeOH Methanol SDA Sabouraud Dextrose Agar SDB Sabouraud Dextrose Broth SLM Sabouraud Liquid Medium TLC Thin layer chromatography U.V. Ultraviolet

CHCl 3 Chloroform

H2SO 4 Sulphuric acid NaCl Sodium chloride

NaH 2PO 4.2H 2O Sodium phosphate (monobasic)

Na 2HPO 4 Sodium phosphate (dibasic anhydrous)

Pharmacognostic evaluation/standardization of drugs Chemical analysis/Colour reactions 1. Formaldehyde-sulfuric acid test:- Reagent :-To four volumes of sulfuric acid add six volumes of formaldehyde solution with stirring and adequate cooling. When the reagent is warm it remains clear for 1 hour. If turbidity developed, this may dispelled by heating in a water bath at 100 c for about one minute. Method :-mix the sample with the reagent and heat at 100 c for 1 min. 2. Forest reagent test:- Reagent:-Mix together equal volumes of a 0.20% (W/V) solution of potassium dichromate, a 30% (v/v) solution of Sulfuric acid, a 20% (w/w) solution of perchloric acid and a 50% (v/v) solution of nitric acid. Method:-Dissolve the sample in a minimum volume of 2M hydrochloric acid and add an equal volume of the reagent. 3. FPN reagent test:- Reagent: - Mix together 5ml of 5 %( w/v) ferric chloride solution, 45ml of a 20% (w/w) solution of perchloric acid and 50ml of a 50% (v/v) solution of nitric acid. Method: - Dissolve the sample in a minimum volume of 2M hydrochloric acid and add an equal volume of the reagent. 4. Liebermann`s reagent test:- Reagent: - Add 1g of sodium or potassium nitrite to 10 ml of sulfuric acid with cooling and swirling to absorb the brown fumes. Method: - Add two or three drops of the reagent to the sample on a white tile. Occasionally it is necessary to carry out the test in a tube and heat in a water bath at 100 c. Many substances gives color with sulfuric acid alone and the test should be repeated using sulfuric acid instead of the reagent. 5. Marquis test:- Reagent: - Carefully mix 100ml of concentrated sulfuric acid with 1 ml of 40% (v/v) formaldehyde solution. Method: - Add a drop of the reagent to the sample on a white tile.

6. Methanol-Potassium hydroxide test:- Reagent: - A 20% (w/v) solution of potassium hydroxide in methanol. Method: - Add a few drops of the reagent to a solution of the sample in methanol and heat it if necessary to boiling point to develop the colour. 7. Sodium picrate test (Steyn test):- Reagent: - Prepare a solution of 5g of sodium bicarbonate Method: - Mix the sample with a few drops of chloroform and sulfuric acid to hasten the reaction while holding a piece of filter paper, impregnated with the reagent, in the vapours that issue from the tube, and heating the contents to 30 c. 8. Nitrous acid test: - Method : - Dissolve the sample in a minimum volume of water and add an amount of solid sodium nitrite equal in volume to the sample followed by a few drops of 2M hydrochloric acid. 9. Vanillin test:- Reagent: - Dissolve 1g of vanillin in sulfuric acid, warming if necessary. Method: - Add two drops of the reagent to the sample, heat in a water bath at 100 c for 30 sec and note any colour that is produced. Dilute the cooled mixture by adding a few drops of water and note any change of colour. 10. Potassium dichromate test:- Method: - Dissolve the sample by shaking in 0.5ml of 2M hydrochloric acid and add a few crystals of potassium dichromate. 11. Aromaticity test:- Method: - Add two or three drops of nitric acid to the sample, heat in a water bath at 100 c for 1 minute, cool the mixture, dilute three to four times with water and make the solution alkaline by the addition of 40% (w/v) solution of sodium hydroxide. 12. Copper sulphate test for sulfonamides:- Method: - Dissolve the sample in a minimum volume of 0.1 %( w/v) sodium hydroxide and add a 1% (w/v) solution of copper sulfate, drop by drop, until the colour change is complete.

13. Ferric chloride test:- Reagent: - Dissolve 5g of anhydrous ferric chloride, or 8.25g of ferric chloride hexahydrate, in 100ml of distilled water. Method: - Add ferric chloride solution to the sample or an ethanolic solution of the sample. 14. Dragendroff reagent:- Reagent: - Dissolve 1g of bismuth subnitrate in 3ml of 10 M hydrochloric acid with the aid of heat. Dilute to 20 ml with water and dissolve 1g of potassium iodide in the mixture. If black bismuth triiodide separates, add 2M hydrochloric acid and more potassium iodide to dissolve it. 15. Folin -Ciocalteu reagent :- Reagent : -Folin-ciocalteu reagent, (Merck KGaA, 64271 Darmstadt, Germany). Method : - Add the diluted reagent to the sample and make the sample alkaline with 2M sodium hydroxide. 16. Mc Nally’s test: - Reagent : - A 0.5% solution of copper sulfate in 10% acetic acid. A freshly prepared 2% (w/v) solution of sodium nitrite. Method: - Dissolve the sample in a few drops of acetone, and add 1 to 2 ml of water. Add three drops of solution 1 and an equal volume of solution 2. Shake and heat in a water bath at 100 c for 3 minutes.

Powder drug study and preparation All dried plant materials were cleaned and cut into small pieces separately. Then each sample was ground in a grinder separately to get powder of the material and was sieved with the help of cotton cloth. For each sample separate piece of cloth was used just to avoid contamination. Chromatographic methods The following solvent systems were applied for TLC: 1. Ethyl acetate-Methanol-Water (100:16.5:13.5)

2. Chloroform-Methanol-Water (80:20:2)

Thin Layer Chromatography (TLC) TLC has been used for biological bioassays, to determine the methods to separate constituents and to test fractions after a column. For separation on normal phase, precoated silica-gel 60F 254 aluminium sheets (Merck) or glass were used. They were developed in conventional TLC chambers. The mobile phases were as ethylacetate- methanol-water (100:16.5:13.5) for lipophilic constituents and chloroform-methanol- water (80:20:2) for hydrophilic constituents. Detection of the compounds was realized by fluorescence extinction under UV light at 254 nm, fluorescence under UV light at 366 nm.

Pharmacological analysis Antioxidant activity A). DPPH radical scavenging assay For the quantitative assay, the Sarker et al method modified was used the stock solution of crude extracts or fractions was prepared using Methanol to achieve a concentration of 10 mg/ml, whereas that for the test compounds and positive standard is prepared at a concentration of 0.5 mg/ml. Dilutions are made to obtain concentrations of 5×10 2, 5×10 3, 5×10 4, 5×10 5, 5×10 6, 5×10 7, 5×10 8, 5×10 9, 5×10 10 mg/ml. Diluted solutions (1.00mL each) are mixed with DPPH (1.00 mL) and allowed to stand for 30 min for any reaction to take place. The UV absorbances of these solutions were recorded at 517 nm. The experiments were performed in triplicate and the average absorption was noted for each concentration. The same procedure is followed for the standard (ascorbic acid) (Sarker et al ., 2006). B). ABTS radical scavenging assay ABTS 2mM solution was prepared by dissolving ABTS (54.8 mg) in distilled water (50ml) and potassium persulphate (17 mM, 0.3 ml) was added. The reaction mixture was left to stand at room temperature overnight in dark before use. To 0.2 ml of various concentrations of both concentration of extracts, DMSO (1.0 ml ) and ABTS solution (0.16 ml) was added to make a final volume of 1.36 ml. Absorbance was measured spectrophotometrically, after 20 minutes at 734nm (Dorman et al ., 2003; Jayaprakasha et al .,2004; Lo and Cheung, 2005).

C). Nitric oxide scavenging assay Nitric oxide scavenging activity was measured spectrophotometrically (Govindarajan et al ., 2003) by mixing sodium nitroprusside (5 mM) dissolved in phosphate buffered saline with different concentrations of methanolic extract (50 –100 µg/ml). After ambient incubation for 30 min, 1.5 ml of this solution was taken and diluted with 1.5 ml of Griess reagent (prepared by dissolving 1% sulphanilamide, 2% phosphoric acid and 0.1% N-1- naphthylethylenediamine dihydrochloride). During reaction, diazotization of nitrite with sulphanilamide and subsequent coupling with N-1-naphthylethylene diamine dihydrochloride resulted in the formation of active chromophore that was estimated at 546 nm and measured percent scavenging activity was compared with ascorbic acid used as standard.

Anti-inflammatory activity 1. Formalin test Swiss albino mice (25-30gm) were divided into different groups of 5 animals each. They were injected with 20µl of 2% Formalin in the ventral surface of right hind paw and the left hind paw was injected with an equal volume of normal saline. Two distinct phase of intensive licking and biting of right hind paw were observed during 0-10 minutes (early phase or neurogenic phase) and 10-30 minutes (late phase or inflammatory phase) after formalin injection. These phases were scored separately for studying drug effect. Vehicle or drugs were administered orally 30 minutes before formalin injection. The time that they spent licking the injected paw was recorded and considered as indicative of nociception (Modified method of Hunskaar and Hole, 1987). 2. Rat paw edema test Rats were divided in to five groups (n= 5). The first group received vehicle only and served as a control, group two, three and four was treated with crude extracts (100, 300 and 500mg/kg). The fifth group treated with aspirin 300 mg/kg. A mark was made on both the hind paws just below the tibiotarsal junction. Readings were recoreded by analogue vernier callipar in cm. The increase in the paw volume was measured in control, standard and sample treated groups for four hour after carrageenan. Percent of inhibition of edema is calculated by % = (Vc-Vt)/Vc x100, where Vt and Vc are the mean relative

49 changes in the paw volume of the test and control respectively (Winter et al ., 1962; Liu et al ., 2005; Vishnukanta and Rana, 2008).

Analgesic activity 1. Hot plate test For hot plate analgesic activity mice were divided into 6 groups (i.e. Group-1 for control, Group-2 and Group-3 and Group 4 for 100 mg/kg, 300mg/kg and 500mg/kg oral doses of crude extract respectively, and Group-D for standard). Each group comprised of 5 animals, weighing 25-30 gm. Acetyl salicylic acid (Aspirin) as 300mg/kg orally was used as the reference compound. The crude drug and the acetyl salicylic acid were diluted in distilled water and administered orally. The control animals were treated orally with the same volume of saline as the crude extract (Dharmasiri et al ., 2003). 2. Acetic acid induce writhing test The test was performed according to the modified method of Koster et al. (1959). Mice were used as the test animals in this method. According to this method writhes were induced by intra peritonial administration of the acetic acid solution 10ml/kg. Thirty minutes prior to the administration of the acetic acid, the animals were treated orally with test substance. Number of writhes was counted for 30 minutes immediately after acetic acid administration. A reduction in the number of writhing as compared to the control animals were considered as evidence for the presence of analgesia and expressed as percent inhibition of writhing. Mice were divided into five groups (i.e. Group 1 for Control, Group 2, 3 and 4 for crude extract 100 mg/kg, 300 mg/kg and 500 mg/kg respectively and group 5 for standard drug aspirin. Each group comprised of 5 animals, weighing 25-35 gm. The crude drug and the standard were diluted with distilled water and administered orally. The control animals were treated orally with the same volume of saline as the crude extract. 3. Tail flick or tail immersion test The test was performed according to the method described by Owoyele et al . 2004. Mice were treated orally with 100, 300 and 500 mg/kg of crude extract. Aspirin 300 mg/kg was used as a standard drug and control group only received normal saline solution. Water

50 was heated to 51± 1°C in a water bath. The time taken for the animal to remove it tails out of the water was recorded after the 30 min. of drug administration. Percentage of response was calculated in comparison with control.

Diuretic activity Modified method of Hook et al ., (1993) was used to screen the diuretic activity of R. fruticosus and V. thapsus different extracts. Albino mice of weights ranging from 25 to 30 g were starved overnight before each test, but allowed free access to water (Hill and Randal, 1976; Sim and Hopcroft, 1976). On the morning of each test, the mice were divided into five groups (3 mice per group). Three groups were orally dosed 100, 300 and 500mg/kg of the tested crude drug. Fourth group received tap water as negative control and fifth group received standard drug Hydrochlorothiazide 10mg/kg body weight. After dosing, each group of mice was placed on wire-mesh secured in a large plastic funnel. The urine was collected over a period of 3 hours. Tests were performed in triplicate. Results are expressed as volumes in ml and diuretic index were calculated.

Gross behavioural activity For monitoring the effects of crude extract on central nervous system, the following procedure was adopted as described by Irwin et al. (1968) and Debprasad et al. (2003). Material and Methods Two group of albino mice of positive NMRI strain of either sex (group comprises of 3 + control) and same weight (i.e. 25-30 g) were selected for neuropharmacological activity experiments. The animals were purchased from HEJ Research Institute of Chemistry, University of Karachi. Animals used for CNS activity were acclimatized first for at least 5 days in the laboratory environment with 12 hours light and 12 hours dark schedule. Animals were housed in standard metal cages and provided food and water ad-libitum .

Preparation of test material Method of obtaining the water fraction and methanol fraction is already described in experimental part of this thesis. The dose of methanol extract was prepared in 0.5 ml distilled water i.e. 300 mg/kg and 500 mg/kg/0.5 ml.

Behavioural Observations The observations of animal (albino mice of 25-30g) treated with the extract of plant showed interesting results of increase or decrease nature in behaviour on comparison with control. The results are given in Table. After treatment with different doses of crude extract, the animals were observed for a total period of 24 hours. The behaviour was recorded according to a modified version of the procedure described by Irwin in 1968 for the following behavioural activities. i) Grooming It means the movements exhibited by the animals. Two groups of three animals each were used for observing changes in grooming response after administration of different extracts and fractions. Group 1 was treated with extract and group 2 was given vehicle only. Each group was observed between 20-120 minutes. ii) Vocalization Means various sounds exhibited by the animals. Two groups of 3 animals were used for each extract. Group 1 was treated with extracts and group 2 was given vehicle only. iii) Irritability It means feeling of restlessness. Same process, dose and duration were used as described above. iv) Passivity Means animals is not active. Same process, dose regime and duration were used as described above. v) Catatonia It is a type of schizophrenia characterized by immobility. Same procedure was used as described above. vi) Spontaneous Activity For observing the increase or decrease in spontaneous activity, same procedure was adopted as given above. Same dose regime and duration was used as described above. vii) Touch Response For observing the increase or decrease in the touch response, same process, dose regime and duration was used as described for other behaviour activities above.

52 viii) Straub Tail It means erection of tail. Group 1 was treated with different extracts and group 2 was given vehicle only. Same process, dose regime and duration were used as described above. ix) Tremor It means shaking of the body. Two groups of 3 animals were used for each extract. Group 1 was treated with different extracts and group 2 was given vehicle only. x) Twitches Slight muscular contraction is called twitches. Dose regime process and same period of observation was used as described above. xi) Convulsion It is a temporary loss of consciousness with severe muscles contraction. Same dose regime and process was used as described above. Each group was observed for 20 minutes and the number of animals showing convulsions was counted. xii) Writhing Means twist or coil violently in pain. For observing this activity same dose regime and procedure was adopted as described above. xiii) Staggering Gait It means walking abruptly. 20-120 minutes observations were made after injecting the dose specified above. xiv) Righting Reflex It is the ability to move in opposite direction during sleeping. Same procedure, dose regime and duration were used as mentioned above. xv) Body Tone It means tightness of the muscles. For observing this activity same dose regime and procedure was adopted as described before. xvi) Grip Strength The animals are observed whether they hold the rod or not. The observations were made for 20 minutes. Method and doses were same as above.

53 xvii) Salivation It is the excess secretion of the saliva. The observations were made for 20-120 minutes. Method and doses were same as above. xviii) Elevation It means rising of animal. The observations were made for 20-120 minutes. Method and doses were same as above. xix) Lacrimation It means secretion of tears from the eyes. After injecting the doses of different extracts the animals were observed visually for lacrimation. The method adopted was the same as mentioned before. xx) Tail Erection It means tail becoming upright and rigid. Doses were used as described above and animals were observed between 20-120 minutes. xxi) Aggressiveness It means restlessness and mental illness. The animals were observed between 20-120 minutes for their aggressiveness after injecting the extracts. xxii) Lethargic It means feeling of drowsiness. Procedure, dose regime and time of observation used were remained the same as described above. xxiii) Thirst It means desire to drink water. The animals were observed between 20-120 minutes for their thirst whether increased or decreased after injecting the extracts.

Assessment of neuropharmacological activity CNS activity was studied by open field test, traction test, head dip test, rearing test, and swimming induced depression test. All the CNS related tests were performed in a calm and peaceful environment. In each test, animals were divided into 5 groups (i.e. Group-A for control, Group-B, Group-C and Group-D for 100mg/kg, 300mg/kg and 500mg/kg oral doses of crude extract respectively, and Group-D for standard). Each group comprised of 5 animals. Diazepam as 2mg/kg orally was used as standard. The crude drug and the diazepam were

54 diluted in distilled water and administered orally. The control animals were treated orally with the same volume of saline as the crude extract. In all the tests observations were made after 30 to 40 minutes of oral dose of the test substance. 1. Open Field Activity The open field apparatus designed in the laboratory consists of 76 X 76 cm square area with opaque walls 42 cm high. The floor is divided by lines into 25 equal squares. Mice weighing 25 to 30gm were used as the test animals in this method. Testing was performed in a quite room under white light as described by Kennett et al., (1985a, b) and Turner, 1965. Animals taken out from their home cages and were placed in the center square of the open field (one at a time). Number of Squares crossed with all four paws was counted for 30 minutes. Activities of control mice and drug treated mice were monitored in a balanced design to avoid order effect. 2. Head Dip Test It is an exploratory test. A specially designed square shaped Head dip box having three holes in each side was used in this study. The observation was to count the number of head dips by the animal through these holes in specified time (Sanchez-Mateo et al. 2002; Kasture et al. , 2002 and Debprasad et al. , 2003). The head dip box in our laboratory is designed for mice. Mice weighing 25 to 30 gm were used in this test. The control and drug treated animals were placed individually in the head dip box and the observations were made for 30 minutes. 3. Cage Crossing Movement The test performed on mice in a specifically designed having rectangular shape. Both control and treated mice were placed in to the cage and their cage crossing movements were noted in 30 minutes. The test is important for the motor activity of animal. This test was performed according to the method described by Crestani et al. (2000). 4. Rearing Test Rearing is also an exploratory behavior test. Mice weighing 25 to 30 gm were used as the test animals. A 1000 ml glass beaker lined with white paper on bottom was used in this study. The observation was count the upward movements of the animal locating the body in an erect position in the beaker (Sakina et al. 1990; Kasture et al. 2002; Sanchez-Mateo et al. 2002). The observations were made for 30 minutes.

5. Traction Test Mice were used as the test animal in this method. Animal were weighing 25 to 30 gm. The observation was to determine the time taken by the animal to travel an iron rod of one-meter length. Firstly mice were trained to make them able to walk on iron rod. Any increase or decrease in time taken by the drug treated animals from control animals to travel the rod describes the sedative or stimulant activity of the drug respectively (Sanchez-Mateo et al. , 2002; Kasture et al ., 2002; Debprasad et al. , 2003). 6. Forced induced Swimming Test Forced induced swimming test was performed according to Sanchez et al. (2002) and Turner (1965). This test determines the muscle and CNS activity of the crude extract. Mice weighing 25 to 30 gm were placed individually for six minutes in the glass tub filled with water at room temperature up to the marked level. Mouse when placed in water suddenly starts to move its front and hind paws. The activity time of animal is determined with the help of stop watch out of total observation time of six minutes.

Evaluation of mechanism of action of drugs Antibacterial activity Test Organisms Antibacterial activity was carried out against Staphylococcus aureus, Escherichia coli and Pseudomona aeruginosa , Shigella flexneri, Citrobacter and Yersenia aldovae. The culture of organisms was maintained on stock culture agar and from the stock culture; a loop full of the culture was inoculated in nutrient broth. The broth were incubated at 37 1 C for twenty-four hours. Inocula were prepared by diluting twenty-four hours old cultures in saline. A dilution of 1:100 was used in all the tests.

Antibacterial Assay Modified soy agar Petri plates, Bauer et al ., (1966), Gnanamani et al., (2003) and Baqir et al ., (1985), were prepared for testing the antibacterial activity of drugs and crude extracts. 0.1ml of diluted culture was poured on each plate and the plates were dried for thirty minutes at 37 C. Disc of 8 mm diameter were used and soaked with different concentration of drug solutions and standard drugs Ampicillin 1mg and Amoxicillin 1mg.

The discs were placed on plates and incubated for twenty-four hours at 37 C. At the end of incubation period, the inhibition zones were measured. Media Media Used for Assay: Tryptic soy agar (Difco) Soybean-casein digest agar medium (USP) Formula per Liter Bactotryptone 15 g Pancreatic digest of casein U.S.P. Bacto soytone 5 g Papaic digest of soybean meal Sodium chloride 5 g Bacto Agar 15 g pH 7.3 0.2 at 25 C. To hydrate the medium, 40 g of hydrated medium was dissolved in 1000mL of cold distilled water. It was heated to dissolve the medium completely and sterilized in autoclave for 15 minutes at 15 lbs/in 2 pressure (121 C). Media used for Culture Maintenance Stock culture (Difco) Brain heart infusion Dehydrated Formula per Liter Calf brains infusion 200 g Beef heart infusion 250 g Bacto proteose peptone 10 g Bacto dextrose 2 g Sodium chloride 5 g Disodium phosphate 2.5 g pH 7.4 0.2 at 25 C

To hydrate the medium, 50 g of dehydrated medium was dissolved in 1000mL of cold distilled water, heated to boiling to dissolve the medium and sterilized in autoclave for fifteen minutes at 15 lbs/in 2 pressure (121 C). Antifungal activity A study on antifungal activity was carried out against Candida albicans, Saccharomyces cerevisiae, Fusarium solani, T. rubrum, Aspergillus parasiticus, M. phaseolinia . A loop full of culture was inoculated in Sabouraud Dextrose Broth. The broth were incubated at 27 1 C for twenty-four hours. Antifungal Assay Bauer et al ., (1966) modified method is used for assay. Sabouraud Dextrose Agar (SDA) Formula g/liter Mycological peptone 10.0 Glucose 40.0 Agar 15.0 pH 5.6 0.2

Directions: Add 15 g of SDA to 1litre of distilled water to dissolve SDA completely, heat the mixture at least 10 minutes to sterilize the SDA medium and autoclave it at 121 C for 15 minutes. Sabouraud Liquid Medium (SLM) Formula g/liter Pancreatic digest of casein 5.0 Peptic digest of fresh meat 5.0 Glucose 20.0 pH 5.7 0.2 Directions Dissolve 30 g of SLM in 1 liter of distilled water. Later dissolve SLA completely and heat the mixture at least 10 minutes, then sterilize the SLA medium by autoclaving at 121 C for 15 minutes.

Toxicological studies 1. Insecticidal activity Freedom from insect infestation and contamination has become an important consideration in storage of grain and to maintain a high quality product. This is also an issue to herbal medicine, therefore, for the determination of pest in herbal medicine the classical method of analysis is used during experiments (Abbott, 1925; Tabassum et al ., 1997; Collins, 1998). Requirements Test insects ( Tribolium castaneum and Sitophilus oryzea ), volatile organic solvent etc.

2. Antihelmintic activity Sample (plants) collected from District Dir lower Khyber Pukhtun Khwa, their methanolic extracts were used. Earthworms, Lumbricus terrestris , four each were placed in a Petri dish containing either aqueous extract (1, 5, 10, 25, 50, 75 and 100 mg / 2ml.) The worms were observed for their spontaneous motility (paralysis) and evoked responses to pin prick which were scored from 0 to 4. The paralytic score was recorded at different time intervals. Immediately after inhibition of response to pin prick, the worms were placed in fresh water and observed for recovery. Duration required for the final recovery or death was noted. Mean paralytic score were with piperazine and Niclosimide as reference standard (Shivkar and Kumar, 2003).

3. Brine shrimp lethality test (BSLT) Meyer et al., (1982) modified method was used to investigate the cytotoxicity of R. fruticosus and V. thapsus plant methanolic extracts, For this test, the tiny crustacean Artemia salina eggs were purchased from Aquarium market Karachi. These eggs were hatched within 48 hours in artificially marine water with proper oxygenation and lightning overnight. Compounds or extracts are tested at initial concentrations of 10, 100, and 1000 µg per ml distilled water in vials containing 5 mL of brine and 10 brine shrimps in 3 replicates. Survivors are counted after 24 hours, with the aid of a stereoscopic microscope, and LD50 values at the 95% confidence limit are calculated using probit analysis software.

RESULTS

Rubus fruticosus Four types of methanolic extracts from R. fruticosus i.e. fruit (RFF), leaves (RFL), root (RFR) and stem (RFS) were screened for the presence of different chemical constituents. The constituents in R. fruticosus were identified by reactions with various chemical reagents mentioned in the experimental part. These reactions showed us characteristics colours which indicate the presence or absence of various groups of compounds. Aromaticity, polyphenols, flavonoids, carbohydrates, tannins and amino-salicylates were found in samples of R. fruticosus (Table 1a and 1b).

Powder drug studies of R. fruticosus with different chemicals were given in Table 3 for RFL, Table 4 for RFR and Table 5 for RFS. These powders were observed in day light, under UV 254 nm and UV 366 nm.

The fluorescence analysis of R. fruticosus various parts i.e. RFF, RFL, RFR and RFS were given in Table 9. The methanolic extracts were dissolved in various solvents and observed under ordinary light, UV light at 254 nm and 366 nm.

Thin layer chromatography of R. fruticosus was performed in two solvent systems. Table 11 and Figure 1 shows the results of TLC of R. fruticosus in ethylacetate-methanol-water (100:16.5:13.5) and chloroform-methanol-water solvent system (80:20:02). The TLC plates were observed in normal light, under UV light at 254 and 366 nm. The Rf of different spots were measured.

The antioxidant capacity was determined using DPPH radical scavenging, ABTS radical scavenging and nitric oxide scavenging assays. The % DPPH radical scavenging for 5×10 -1 mg/ml of RFF, RFL, RFR and RFS are 96, 92.88, 90.07 and 89.88 respectively (Table 13, Graph 1).

During ABTS radical scavenging assay the % scavenging at dose of 50 and 100 µg/ml were 36.19±1.55, 73.02±0.48 for RFF: 24.06±0.65, 50.01±1.07 for RFL: 21.37±1.21, 43.16±0.94 for RFR and 18.77±1.01, 39.15±1.47 for RFS respectively (Table 15, Graph 1).

Similarly nitric oxide % radical scavenging at dose of 50 µg/ml and 100 µg/ml were 23.21±1.03, 47.05±0.69 for RFF: 19.56±0.88, 38.17±1.29 for RFL: 17.75±1.44, 31.07±0.39 for RFR and 13.26±1.31, 26.32±1.66 for RFS respectively (Table 16, Graph 1).

The results of formalin induced inflammation depicted in Table 17 and Graph 2. R. fruticosus (100, 300 and 500 mg/kg) caused a dose dependent inhibition. The significant inhibitions observed at p≤0.05 were 35±1.39 and 35±0.73; 60.67% and 48.52 % at a dose of 500 mg/kg, for first and second phase respectively for RFF. RFL (55%, 55.88%), RFR (21.34%, 19.11%) and RFS (10%, 11.76%) showed inhibitions at 500 mg/kg for first and second phase, respectively.

The inhibitory activity of R. fruticosus extracts against Carrageenan induced paw edema was presented in Table 18 and Graph 3. The results were measured in mm and percent inhibition were calculated for four parts (RFF, RFL, RFF and RFS) of R. fruticosus . Aspirin, as a standard at dose of 300 mg/kg markedly reduced the edema (25.59%; 24%; 33.46% respectively).

The results of hot plate test of R. fruticosus were given in Table 19 and Graph 4 at three different doses for each part. The dose dependant increase in the latency time was observed at 100, 300 and 500 mg/kg at p ≤0.05. The result s were significant at 300 and 500 mg/kg for RFF. RFL has significant activity at 500 mg/kg. Similarly the response was significant for higher doses in RFR and RFS.

R. fruticosus at 100, 300 and 500 mg/kg caused an inhibition of the writhing response. RFF showed 61.06% (first phase) and 63.26% (second phase) inhibition at 500 mg/kg

61 dose. RFL showed about 79.73% and 77.55% inhibition at both 1st and 2 nd phase respectively at 500 mg/kg dose. RFR 60.93%, 48.57% while RFS 21.86%, 19.97% at 500 mg/kg dose for first and second phase respectively (Table 21, Graph 5). Animals from the control group showed 75±4.36 (first phase) and 49± 2.81 (second phase) abdominal writhing accumulated at 30 min after acetic acid injection.

The tail-flick method was also used to evaluate the analgesic effect of the R. fruticosus different parts methanolic extracts. Albinos were treated with 100, 300 and 500 mg/kg of extracts. 10ml normal saline per kg was administered to control group. Both significant and non-significant results for RFF, RFL, RFR and RFS are given in Table 22 and Graph 6.

Diuretic activity was determined after a period of 3 hours. The urine of control group was 0.06±0.187 ml. The diuretic indexes of the treated mice were calculated for RFF the value came out to be 6, 7.33 and 4.93 for the 100, 300 and 500 mg/kg of doses respectively. The indexes calculated were 0.5, 4.66 and 0.66 for RFL, 0.33, 0.33 and 1.33 for RFR, 3.66, 4.16 and 0.66 for RFS. The RFF showed significant results. Hydrochlorothiazide was used as standard showed diuretic index 8.5 (Table 24).

Table 25, 26 and 27 showed results of gross behaviour activity of RFF at 100, 300 and 500 mg/kg in mice respectively. RFF increased the motor functions in open-field test and decreased in head dip test (Table 49, Graph 7) in comparison with negative control group. For 100, 300 and 500 mg/kg of RFF open field in terms of readings are 474.2±2.44, 404 ±1.66 and 435 ± 1.94 while head dip 34.8±2.48, 26.6 ± 2.92 and 16.4 ± 2.28 respectively. The control showed 287±2.62 for open field and 43.8 + 3.90 for head dip at p≤0.05 . The results were compared with Diazepam 2 mg/kg, Imipramine 15 mg/kg and Caffeine 15 mg/kg.

Table 28, 29 and 30 showed results of gross behaviour activity of RFL at 100, 300 and 500 mg/kg in mice respectively. RFL increased the motor functions in open-field test and decreased in head dip activity (Table 49, Graph 7) in comparison with negative control

62 group. For 100, 300 and 500 mg/kg of RFL open field in terms of readings are 255.4±2.74, 259.4 ± 1.29 and 306.2± 2.54 while head dip 11.8±2.32, 17.6 ± 1.97 and 15.4 ± 2.19 respectively.

Table 31, 32 and 33 showed results of gross behaviour activity of RFR at 100, 300 and 500 mg/kg in mice respectively. RFR increased the motor functions in open-field test and decreased in head dip activity (Table 49, Graph 7) in comparison with negative control group. For 100, 300 and 500 mg/kg of RFR open field in terms of readings are 422.2±0.58, 358.4± 2.21 and 306.6 ± 1.92 while head dip 14.6±1.73, 9.6±1.94 and 11 ± 1.79, respectively.

Table 34, 35 and 36 showed results of gross behaviour activity of RFS at 100, 300 and 500 mg/kg in mice respectively. RFS increased the motor functions in open-field test and decreased in head dip activity (Table 49, Graph 7) in comparison with negative control group. For 100, 300 and 500 mg/kg of RFS open field in terms of readings are 293.8±1.47, 197.6 ± 1.51 and 186.8 ± 2.09 while head dip 33.2 ± 0.86, 19 ± 1.27 and 32.4 ± 1.21, respectively.

In the cage cross locomotor activity were recorded in terms of numbers of squares crossed by treated and untreated mice. So activity observed for control group was 65.4 ±4.13. For RFF treated animals at dose of 100, 300 and 500 mg/kg, the means ±S.E.M. of crossings number were 65.8±2.81, 70±1.71 and 90.6±1.64, respectively (Table 51, Graph 8). Similarly for RFL (44.6±1.87, 62.6 ± 2.12, 71.4 ±1.40), RFR (56.4±1.17, 52.2 ± 1.86, 46.2 ± 1.78) and RFS (65.8±2.34, 50.2±2.18, 82.2±1.94) the means ±S.E.M. of crossings were found at the same dose pattern as for RFF.

The exploratory rearing activity observed for control group was 50.6 ± 1.53. For RFF treated animals at dose of 100, 300 and 500 mg/kg, the means ±S.E.M. of crossings number were 34±1.21, 46±1.43 and 55.4±1.61, respectively (Table 51, Graph 8). Similarly for RFL 28±1.51, 41±1.11, 57.5 ± 0.81, RFR 23.5±1.15, 33.8± 0.81, 18.6 ± 1.43 and RFS 24±1.42, 36.4±1.13, 45.4±0.83 the means ±S.E.M. of crossings were found

63 at the same dose pattern as for RFF at p≤0.05. The results were compared with standard drugs Diazepam 2 mg/kg, Imipramine 15 mg/kg and Caffeine 15 mg/kg.

The results of motor co-ordination activity on traction test were noted for 4 and 1/2 hours. The significant and non-significant differences were given in Table 53. The time for crossing the rod and numbers of fall were observed and compared with control and standard drugs. The data were represented with ± S.E.M. at p≤0.05.

The mobility and immobility time were recorded in forced swimming test. The mean observations ±S.E.M. were presented in Table 55 and Graph 9. The mobility time in minutes for the control group was 3:45 ± 0.059. Motility time for RFF treated groups of mice at dose of 100, 300 and 500 mg/kg were 1:14±0.0323, 0:25 ± 0.062 and 1:25 ± 0.0338 at p≤0.05. Similarly for RFL (2:27±0.059, 2:22 ± 0.076, 2:04 ± 0.028), RFR (1:19±0.0193, 2:07±0.0341, 1:11±0.2016) and RFS (2:28±0.0316, 2:03 ± 0.030, 3:03 ± 0.0353) the motility time (mean) ± S.E.M. were found out at 100, 300 and 500 mg/kg doses at p≤0.05.

The results of antibacterial activity were measured as zone of inhibition in mm against 8 strains of bacteria (E. coli , S. typhi, S. aureus, M. luteus, Citrobacter, B. subtilis, P. aeruginosa and P. mirabilis ) including both gram positive and gram negative. Zone of inhibition with S.E.M. and MIC for RFF are given in Table 57 for RFL in Table 58, for RFR Table 59 and for RFS in Table 60. The zones of inhibition for standard drugs are also given in these tables. The Graph 10 showed the zone of inhibition for standards and samples. The antibacterial activities of various parts of R. fruticosus were compared with standard drugs.

RFS, RFL, RFR and RFS showed no antifungal activity against S. cerevisiae, A. parasiticus, T. rubrum, M. phaseolinia, C. albican, F. solani, A. niger and A. effusus. Zone of inhibition for standard drugs Itraconazole 2 mg and Amphoteracin B 2 mg are given in Table 65.

The insecticidal activity was monitored against two types of insects T. castaneum and S. oryzae at the concentrations of 1, 5, 10, 25, 50, 75 and 100 mg. RFF at 25 mg and 50 mg showed 10% mortality against S. oryzae and T. castaneum (Table 66). The toxicity results of RFL are presented in Table 67. Table 68 and 69 showed the insecticidal activity of RFR and RFS respectively. Permithrin was used as standard at concentration of 235.9 μg/cm 2, showed 100% mortality.

Antihelmintic activity was studied against Lumbricus terrestris , the observed effects of RFF were given in Table 74. The concentration of 5, 10, 25, 50, 75 and 100 mg/kg were applied and compared with standard drugs Niclosimide. Table 75 indicates anthelmintic activity of RFL, similarly Table 76 and 77 for RFR and RFS respectively. The mean paralytic time and mean death time of different doses were recorded.

Brine shrimp lethality test were carried out to evaluate the cytotoxic potential of R. fruticosus various parts. LD 50 114.2281 μg/ml for RFF (Table 83, Graph 11), 44.89 μg/ml for RFL (Table 84, Graph 11), 19.4803 μg/ml for RFR (Table 85, Graph 11) and 90.7237

μg/ml for RFS (Table 86, Graph 11) was determined. Cyclophosphamide (LD50 15.7 µg/ml) was used as standard (Table 91, Graph 11).

Verbascum thapsus The four types of methanolic extracts from V. thapsus i.e. fruit (VTF), leaves (VTL), root (VTR) and stem (VTS) were screened as follows.

The presence of different constituents in V. thapsus was identified by reactions with various chemical reagents. These reactions showed us characteristics colours which indicate the presence or absence of various groups of compounds. We found the presence of aromaticity, carbohydrates, tannins, flavonoids, alkaloids, polyphenols and aminosalicylates in our samples (Table 2a and 2b).

Powder drug studies of V. thapsus with different chemicals were given in Table 6 for VTL, Table 7 for VTR and Table 8 for VTS. These powders were observed in day light, under UV 254 nm, and 366 nm.

The fluorescence analysis of V. thapsus various parts i.e. VTF, VTL, VTR and VTS were given in Table 10. The methanolic extracts were dissolved in various solvents and observed under ordinary light, UV light at 254 nm and 366 nm.

Thin layer chromatography of V. thapsus was performed in two solvent systems. Table 12 and Figure 1 shows the results of TLC of V. thapsus in ethylacetate-methanol-water (100:16.5:13.5) and chloroform-methanol-water solvent system (80:20:02). The TLC plates were observed in normal light, under UV light at 254 and 366 nm. The Rf of all spots were measured.

The antioxidant capacity was determined using DPPH radical scavenging, ABTS radical scavenging and nitric oxide radical scavenging assays. The % radical scavenging for 5×10 -1 mg/ml of VTF, VTL, VTR and VTS are 89.40, 87.73, 96.13 and 85.05 respectively (Table 14, Graph 1).

During ABTS radical scavenging assay the % scavenging at dose of 50 and 100 µg/ml were 29.25±1.30, 62.28±0.83 for VTF: 26.50±1.28, 54.42±1.02 for VTL: 21.62±1.35, 45.97±1.62 for VTR and 17.23±1.62, 35.91±1.07 for VTS respectively (Table 15, Graph 1).

Similarly nitric oxide % radical scavenging at dose of 50 and 100 µg/ml was 17.08±1.19, 32.99±1.87 for VTF: 14.80±1.52, 26.67±1.23 for VTL: 11.05±1.35, 19.63±1.62 for VTR and 9.61±1.08, 12.32±1.96 for VTS respectively (Table 16, Graph 1). Ascorbic acid was used as standard.

The results of formalin induced inflammation illustrated in Table 17 and Graph 2 showed that the V. thapsus (100, 300 and 500 mg/kg) caused a dose dependent inhibition. The

66 significant inhibitions were observed at p≤0.05: 56.17%, 34.48% at a dose of 500 mg/kg, for first and second phase respectively for VTF. VTL (50.56%, 48.52%), VTR (38.20%, 36.76%) and VTS (22.47%, 17.46%) showed inhibitions at 500 mg/kg for first and second phase, respectively.

The inhibitory activity of V. thapsus extracts against Carrageenan induced paw edema was presented in Table 18, Graph 3. The results were measured in mm and percent inhibition were calculated for four parts (VTF, VTL, VTF and VTS) of V. thapsus . Aspirin, as a standard at dose of 300 mg/kg markedly reduced the edema (25.59%; 24%; 33.46% respectively).

The results of hot plate test of V. thapsus were given in Table 20 and Graph 4 at three different doses for each part. The dose dependant increase in the latency time was observed at 100, 300 and 500 mg/kg at p≤0.05. The results were significant at 300 and 500 mg/kg for VTF. VTL has significant activity at 500 mg/kg. Similarly the response was significant for higher doses in VTR and RFS (Graph 4).

V. thapsus at 100, 300 and 500 mg/kg caused an inhibition of the writhing response. VTF showed 73.73% (first phase) and 62.85% (second phase) inhibition at 500 mg/kg dose. VTL showed 64% for first phase 58.57% for second phase inhibition at 500 mg/kg dose. VTR 36.93%, 47.95% while VTS 33.46%, 36.12% at 500 mg/kg dose for first and second phase respectively. The results are presented in Table 21 and Graph 5. Animals from the control group showed 75±4.36 (first phase) and 49± 2.81 (second phase) abdominal writhing accumulated at 30 min after acetic acid injection.

The tail-flick method was also used to evaluate the analgesic effect of the V. thapsus different parts methanolic extracts. Albinos were treated with 100, 300 and 500 mg/kg of extracts. 10ml normal saline per kg was administered to control group. Both significant and non-significant results for VTF, VTL, VTR and VTS are given in Table 23 and Graph 6).

Diuretic activity was determined for a period of 3 hours. The urine of control group was 0.06±0.187 ml. The diuretic indexes of the treated mice were calculated for VTF the value came out to be 3.66, 4.16 and 0.66 for the 100, 300 and 500 mg/kg of doses respectively. The indexes calculated were 1, 0.33 and 0.66 for VTL, 0.83, 0.83 and 1 for VTR, 1.33, 0.5 and 2 for VTS. The VTF showed significant results at a dose of 100 mg/kg. Hydrochlorothiazide was used as standard showed diuretic index 8.5 (Table 24).

Table 37, 38 and 39 showed results of gross behaviour activity of VTF at 100, 300 and 500 mg/kg in mice respectively. VTF increased the motor functions in the open-field test and decreased in head dip activity (Table 50, Graph 7) in comparison with negative control group. For 100, 300 and 500 mg/kg of VTF open field in terms of readings are 298.2 ± 3.82, 322.6 ± 2.76 and 361.8±2.32 while head dip 22.4 ± 3.71, 16.2 ± 2.66 and 8.2±2.48 respectively. The negative control group activity was 287±2.62 for open field and 43.8 + 3.90 for head dip at p≤0.05 . The results were compared with standard drugs Diazepam 2 mg/kg, Imipramine 15 mg/kg and Caffeine 15 mg/kg.

Table 40, 41 and 42 showed results of gross behaviour activity of VTL at 100, 300 and 500 mg/kg in mice respectively. VTL decreased the motor functions in the open-field test and also in head dip activity (Table 50, Graph 7) in comparison with negative control group. For 100, 300 and 500 mg/kg of VTL open field in terms of readings are 108±2.52, 114 ± 2.60 and 117± 1.87 while head dip 18±2.63, 10.4 ± 2.86 and 9.2 ±1.99 respectively.

Table 43, 44 and 45 showed results of gross behaviour activity of VTR at 100, 300 and 500 mg/kg in mice respectively. VTR increased the motor functions in the open-field test and also in head dip test (Table 50, Graph 7) in comparison with negative control group. For 100, 300 and 500 mg/kg of VTR open field in terms of readings are 156±2.85, 471.8± 3.19 and 475 ± 1.52 while head dip 26.6±2.78, 21.2±2.18 and 44.2 ± 1.50 respectively.

Table 46, 47 and 48 showed results of gross behaviour activity of VTS at 100, 300 and 500 mg/kg in mice respectively. VTS increased the motor functions in the open-field test and decreased in head dip activity (Table 50, Graph 7) in comparison with negative control group. For 100, 300 and 500 mg/kg of VTS open field in terms of readings are 498.4±2.55, 490.6 ± 2.81 and 469.4 ±2.63 ± 2.09 while head dip 27.8 ± 2.93, 27.4 ± 0.93 and 23.2 + 2.75 respectively.

In the cage cross motor activity were recorded in terms of numbers of squares crossed by treated and untreated mice. The activity observed for control group was 65.4 ±4.13. The results for VTF treated animals at dose of 100, 300 and 500 mg/kg were recorded. The means ±S.E.M. of crossings were 42.8±0.97, 57±0.90 and 40.2±0.67, respectively (Table 52, Graph 8). Similarly for VTL (11.2±0.38, 39.2 ± 1.78, 59.2 ±1.69), VTR (26.6±0.93, 21.1 ± 0.86, 44.2 ± 1.50) and VTS (78±0.95, 74.8±1.60, 55.2±0.49) the means ±S.E.M. of crossings were found at the same dose pattern as for VTF.

The exploratory rearing activity observed for control group was 50.6 ± 1.53. For VTF treated animals at dose of 100, 300 and 500 mg/kg, the means ±S.E.M. of crossings were 14±0.81, 19.4±1.43 and 16±1.10, respectively (Table 52, Graph 8). Similarly for VTL (07±0.51, 19±1.21, 26.4 ± 1.21), VTR (18±1.3, 13.8± 0.76, 19.6 ± 1.03) and VTS (24±1.12, 19.4±1.33, 14.4±1.03) the means ±S.E.M. of crossings were found at the same dose pattern as for VTF at p≤0.05. The results in open field, head dip, cage cross and rearing activities were compared with standard drugs Diazepam 2 mg/kg, Imipramine 15 mg/kg and Caffeine 15 mg/kg.

The results of motor co-ordination activity on traction test were noted for 4 and 1/2 hours. The significance and non significant differences were given in table 54. The time for crossing the rod and numbers of fall were observed and compared with control and standard drugs. The data were represented with ± S.E.M. at p≤0.05.

The mobility and immobility time were recoded in forced swimming test. The mean observations ±S.E.M. were presented in Table 56 and Graph 9. The mobility time in

69 minutes for the control group was 3:45 ± 0.059. Motility time for VTF treated groups of mice at dose of 100, 300 and 500 mg/kg were 1:36±0.0285, 2:09 ± 0.0336 and 1:40 ± 0.0296 at p≤0.05. Similarly for VTL (1:30±0.0236, 0:36 ± 0.0283, 2:20 ± 0.0296), VTR (2:40±0.0296, 3:07±0.0418, 2:58±0.0273) and VTS (2:51±0.0303, 2:39 ± 0.026, 3:34 ± 0.0238) the motility time (mean) ± S.E.M. were found out at 100, 300 and 500 mg/kg doses at p≤0.05.

The results of antibacterial activity were measured as zone of inhibition in mm against 8 species of bacteria including both gram positive and gram negative. Zone of inhibition with S.E.M. and MIC for VTF are given in Table 61 for VTL in Table 62, for VTR Table 63 and for VTS in Table 64. The zones of inhibition for standard drugs are also given in these tables. The Graph 10 showed the zone of inhibition for standards and samples. The antibacterial activity of V. thapsus different parts was compared with standard drugs.

VTF, VTL, VTR and VTS showed no antifungal activity against S. cerevisiae, A. parasiticus, T. rubrum, M. phaseolinia, C. albican, F. solani, A. niger and A. effusus. Zone of inhibition for standard drugs Itraconazole 2 mg and Amphoteracin B 2 mg are given in Table 65.

The insecticidal activity was monitored against two types of insects T. castaneum and S. oryzae at concentrations of 1, 5, 10, 25, 50, 75 and 100 mg. VTF at 100mg showed 10% mortality against S. oryzae and 0% mortality against T. castaneum (Table 70). The toxicity results of VTL are presented in Table 71. Table 72 and 73 showed the insecticidal activity of VTR and VTS respectively. Permithrin was used as standard at concentration of 235.9 μg/cm 2, showed 100% mortality.

Antihelmintic activity was studied against Lumbricus terrestris , the observed effects of VTF were given in Table 78. The concentration of 5, 10, 25, 50, 75 and 100 mg/kg were applied and compared with standard drugs Niclosimide. Table 79 indicates anthelmintic activity of VTL similarly Table 80 and 81 for VTR and VTS respectively. The mean paralytic time and mean death time of different doses were recorded.

Brine shrimp lethality test were carried out to evaluate the cytotoxic potential of V. thapsus various parts. LD 50 3.0872 μg/ml for VTF (Table 87, Graph 11), 3.4207 μg/ml for VTL (Table 88, Graph 11), 130.995 μg/ml for VTR (Table 89, Graph 11) and 45.728

μg/ml for VTS (Table 90, Graph 11) was determined. Cyclophosphamide (LD50 15.7 µg/ml) was used as standard (Table 91, Graph 11).

Table 1a: An overview of chemical constituent identified by chemical reagents

No. Plant name Chemical Rubus fruticosus (Extracts of different parts) test/r eactions

Fruit Leaves Root Stem 1. Formaldehyde sulfuric acid test Observation(normal Dark brown Green Brown red Brown light Results Benzodiazepines present 2. Forest Reagent test OB Brown orange++ Green Brown orange+++ Brown orange+ Results Phenothiazine 3. FPN Reagent test OB Orange yellow Brown Orange yellow Orange yellow Results Phenothiazines and related groups 4. Liebermann`s reagent test OB Red brown + Brown Red brown++ Red brown +++ Results Benzodiazaphens and phenoxyphenypropionic acid derivatives 5. Marquis test OB Yellow brown Yellow brown Red brown Red brown Results Amphetamines and related groups 6. Methanol-Potassium hydroxide test OB Before heating Greenish yellow Marion Mustard After heating Yellow Brown Orange red Brown pink Results Quinines, dions and phenols derivatives are present 7. Sodium picrate test (Steyn test) OB No colour No colour No colour No colour Results Cyanide -ve -ve -ve -ve 8. Nitrous acid test OB Orange Orange(reddish) Orange Orange+++ Results Sulfonamides

Table 1b: An overview of chemical constituent identified by chemical reagents

No. Plant name Chemical Rubus fruticosus (Extracts of different parts) test/reactions Fruit Leaves Root Stem 9. Vanillin test OB Before dilution Reddish brown Reddish brown Reddish brown Reddish brown After dilution Green Green Green Green Results Barbiturates 10. Potassium dichromate test OB Green brown Green brown Yellow brown Green brown Results Phenol derivatives 11. Aromaticity test OB Acid colour Yellow Yellow Yellow Yellow Colour in base Orange Orange red Orange red Orange red Results Aromatic ring present 12. Copper sulphate test for sulfonamides OB Green Brown Green Green Results +ve +ve +ve +ve 13. Ferric chloride test OB Violet Violet black Green dark Blue violet Results Phenols and salicylates 14. Dragendroff reagent OB Brown orange Brown orange Brown orange Brown orange Results Alkaloidal base present 15. Folin-Ciocalteu reagent OB Blue Blue Blue Blue Results Polyphenolic compounds present 16. Mc Nally’s test OB Brown Greenish Brown Brown brown Results Aminosalicylic acid derivatives

Table 2a: An overview of chemical constituent identified by chemical reagents

S. Plant name No. Chemical Verbascum thapsus (Extracts of different parts) test/ reactions Fruit Leaves Root Stem 1. Formaldehyde sulfuric acid test Observation(OB) Brown red Reddish brown Brown red Dark brown Results Benzodiazepins present 2. Forest Reagent test OB Brown orange Brown orange Brown orange++ Brown orange++ Results Phenothiazine 3. FPN Reagent test OB Orange yellow Brown orange Orange Brownish orange Results Phenothiazine 4. Liebermann`s reagent test OB Orange brown Brown Brown Brown Results Benzodiazaphens and phenoxyphenypropionic acid derivatives 5. Marquis test OB Red brown Red brown Red brown Red brown Results Amphetamines and related groups 6. Methanol-Potassium hydroxide test OB Orange Green yellowish Orange Yellow orange Results Quinines, dions and phenols derivatives are present 7. Sodium picrate test (Steyn test) OB No colour No colour No colour No colour Results Cyanide -ve -ve -ve -ve 8. Nitrous acid test OB Yellow Yellow Yellow Orange Results Sulfonamides

Table 2b: An overview of chemical constituent identified by chemical reagents

No. Plant name Chemical Verbascum thapsus (Extracts of different parts) test/reactions Fruit Leaves Root Stem 9. Vanillin test OB Before dilution Brown orange Brown red Brown orange Brown orange After dilution Brown orange Green Brown orange Brown orange Results Barbiturates 10. Potassium dichromate test OB Green brown Yellow brown Yellow brown Green brown Results Phenolic compounds 11. Aromaticity test OB Acid colour Yellow Yellow Yellow Colourless Colour in base Orange Red Orange Yellow Results Aromatic ring present 12. Copper sulphate test for sulfonamides OB Green Green Green Green Results Sulfonamides present 13. Ferric chloride test OB Yellow brown Green Green yellow Yellow brown Results Phenols and salicylates 14. Dragendroff reagent OB Red orange Brown Red orange Red orange orange Results Alkaloidal base present 15. Folin-Ciocalteu reagent OB Blue Blue Blue Blue Results Polyphenols 16. Mc Nally’s test OB Brown Green Brown Brown Results Aminosalicylic acid derivatives

Table 3: Fluorescence analysis of powder of Rubus fruticosus leaves

S. No. PROTOCOL OBSERVATIONS UNDER Ordinary light UV light 254 UV light 366 01 Powder Green Brown Green Dark brown 02 Powder treated with 1.0 N Black brown Greenish black Black NaOH in MEOH 03 Powder treated with 5% FeCl3 Green Blackish brown Dark brown

04 Powder treated with 50% H 2 SO 4 Brown Light brown Reddish brown

05 Powder treated with 1.0 N HCl Brown Green brown Red brown

06 Powder treated with 50% HNO 3 Green brown Dark green Dark brown

07 Powder treated with 1.0 N NaOH Green brown Brown Green brown in water

Table 4: Fluorescence analysis of powder of Rubus fruticosus root

S. NO. PROTOCOL OBSERVATIONS UNDER

Ordinary light UV light 254 UV light 366 01 Powder Blackish brown Green brown Black brown

02 Powder treated with 1.0 N NaOH Brown orange Green brown Black in MeOH

03 Powder treated with 5% FeCl 3 Brown Dark green Blackish brown

04 Powder treated with 50% H 2SO 4 Light brown Green Greenish 05 Powder treated with 1.0 N HCl Light brown Green Colourless

06 Powder treated with 50% HNO 3 Dark brown Yellow brown Dark Brown 07 Powder treated with 1.0 N NaOH Light brown Green brown Reddish brown in water

Table 5: Fluorescence analysis of powder of Rubus fruticosus stem

S. NO. PROTOCOL OBSERVATIONS UNDER Ordinary light UV light 254 UV light 366 01 Powder Light brown Yellow brown Reddish brown

02 Powder treated with 1.0 N NaOH Brown orange Green brown Black in MeOH 03 Powder treated with 5% FeCl 3 Brown Dark green Blackish brown

04 Powder treated with 50% H 2SO 4 Black Grassy Green Greenish brown

05 Powder treated with 1.0 N HCl Muddy colour Green Colourless

06 Powder treated with 50% HNO 3 Dark brown Yellow brown Dark Brown

07 Powder treated with 1.0 N NaOH Light brown Green brown Black brown in water

Table 6: Fluorescence analysis of powder of Verbascum thapsus leaves

S. NO. PROTOCOL OBSERVATIONS UNDER

Ordinary light UV light 254 UV light 366 01 Powder Brown Greenish brown Dark brown

02 Powder treated with 1.0 N NaOH Orange brown Greenish Reddish brown in MeOH orange 03 Powder treated with 5% FeCl 3 Green Blackish brown Dark brown

04 Powder treated with 50% H 2SO 4 Light green Green Greenish black

05 Powder treated with 1.0 N HCl Green Green brown Red brown

06 Powder treated with 50% HNO 3 Green brown Dark green Dark brown

07 Powder treated with 1.0 N NaOH Light brown Greenish Dark brown in water brown

Table 7: Fluorescence analysis of powder of Verbascum thapsus root

S. NO. PROTOCOL OBSERVATIONS UNDER

Ordinary light UV light 254 UV light 366 01 Powder Blackish brown Green brown Black brown

02 Powder treated with 1.0 N NaOH Brown orange Green brown Black in MeOH 03 Powder treated with 5% FeCl 3 Brown Dark green Blackish brown

04 Powder treated with 50% H 2SO 4 Brown orange Green Black

05 Powder treated with 1.0 N HCl Brown orange Green Black

06 Powder treated with 50% HNO 3 Dark brown Yellow brown Dark Brown

07 Powder treated with 1.0 N NaOH Reddish brown Green brown Reddish brown in water

Table 8: Fluorescence analysis of powder of Verbascum thapsus stem

S. NO. PROTOCOL OBSERVATIONS UNDER

Ordinary light UV light 254 UV light 366 01 Powder Brown Greenish brown Dark brown

02 Powder treated with 1.0 N NaOH Brown orange Green brown Black in MeOH 03 Powder treated with 5% FeCl 3 Brown Dark green Blackish brown

04 Powder treated with 50% H 2SO 4 Brown orange Green Greenish black

05 Powder treated with 1.0 N HCl Brown orange Dark Green Greenish black

06 Powder treated with 50% HNO 3 Dark brown Yellow brown Dark Brown

07 Powder treated with 1.0 N NaOH Light brown Green brown Dark brown in water

Table 9: - Fluorescence analysis of Rubus fruticosus different extracts

Solvents RFL RFR RFS

Visible UV UV Visible UV UV Visible UV UV light 254 nm 366nm light 254 nm 366nm light 254 nm 366nm

Water GB G LG B G LG B G DB

1% H2SO4 BG G BG B G DB GB G DB

5% H2SO4 G G OG B G DB GB G LG

66% H2SO4 G G BG OB DG Bl OB G LG

H2SO4(pure) DG G G RB G LG OB G LB

1%HCl BG G G B G B LGB G DB

5%HCl G G BG B G B GB G LG

Chloroform G G LG RB G LG GB G LG

Ethanol G G VLG B G LG G G LG

Formaldehyde G G VLG OB G LG GB G LG

Nitric acid O G BL OB G Bl OB G B

Diethyl ether I I I B G B GB G RB

Ethyl acetate G G GB B G LB GB G RB

N- hexane G G R I I I BG G LG

Acetone BG G LG B G LG G DG LG

Methanol G G LG B G LG B G LB

RFL, RFR and RFS = Rubus fruticosus leaves, root and stem respectively. B=brown, G= green, LG =light green, YB= yellow brown, GBl= greenish black, DG= dark brown, W=wight, SB= sandy brown,RB=red brown, I= insoluble, Gr= grey, VLG=very light green

Table 10: - Fluorescence analysis of Verbascum thapsus different extracts

Solvents VTL VTR VTS

Visible UV UV Visible UV UV Visible UV UV light 254 nm 366nm light 254 nm 366nm light 254 nm 366nm

Water G G LG B DG LG GB G VLG

1% H2SO4 G G LG B LG LG B G VLG

5% H2SO4 BG G LG B G LG B G VLG

66% H2SO4 G DG B RB DG LG B G VLG

H2SO4(pure) RB G LG RB DG LG RB G LG

1%HCl GB G B SB G G SB G VLG

5%HCl G G LG SB DG LG B G VLG

Chloroform G G LG B G Gr B G LG

Ethanol G G LG B G LG B G LG

Formaldehyde G G LG B DG W B G LG

Nitric acid B G B YB G GBl B G LG

Diethyl ether G G LG I I I B G VLG

Ethyl acetate G G B I I I B G LG

N- hexane I I I I I I I I I

Acetone G G B B G GB B G LG

Methanol G G LG B G LG B G LG

VTL, VTR and VTS = Verbascum thapsus leaves, root and stem respectively. B=brown, G= green, LG =light green, YB= yellow brown, GBl= greenish black, DG= dark brown, W=wight, SB= sandy brown,RB=red brown, I= insoluble, Gr= grey, VLG=very light green

Table 11: Rf values of R. fruticosus extracts by thin layer chromatography (TLC)

Tested Solvent systems Rf value of Rf value of colour extract colo ur spots at spots at 366 nm 254 nm

Fruit Ethyl acetate: Methanol: 0.38, 0.42, 0.66 0.59

Water (100: 16.5: 13.5) 0. 73, 0.81

Chloroform: Methanol: 0.17, 0.266, 0.4, 0.25 0.48, 0.6 Water (80 : 20 : 2)

Leaves Ethyl acetate: Methanol: 0.33, 0.62, 0.64, 0.72 0.75 Water (100: 16.5: 13.5)

Chloroform: Methanol: 0.14, 0.42, 0.53, 0.35, 0.45, 0.72 0.64, 0.8 Water (80 : 20 : 2)

Root Ethyl acetate: Methanol: 0.38, 0.43 -

Water (100: 16.5: 13.5)

Chloroform: Methanol: 0.13, 0.21, 0.46, 0.67 0.6, 0.72, 0.78 Water (80 : 20 : 2)

Stem Ethyl acetate: Methanol: 0.36, 0.43 -

Water (100: 16.5: 13.5) Chloroform: Methanol: 0.16, 0.24, 0.46, 0.21 0.6, 0.72, 0.78 Water (80 : 20 : 2)

Table 12: Rf values of V. thapsus extracts by thin layer chromatography (TLC)

Tested Solvent systems Rf value of color Rf value of color extract spots at 254 nm spots at 366 nm

Fruit Ethyl acetate: Methanol: 0.32, 0.26, 0.46 -

Wa ter (100: 16.5: 13.5) 0.59 , 0.78

Chloroform: Methanol: 0.26, 0.5, 0.7, - 0.76 Water (80 : 20 : 2)

Leaves Ethyl acetate: Methanol: 0.14, 0.22, 0.32 -

Water (100: 16.5: 13.5)

Chloroform: Methanol: 0.53, 0.6, 0.66, - 0.74 Water (80 : 20 : 2)

Root Ethyl acetate: Methanol: 0.36, 0.48, 0.52, 0.46, 0.59 0.78 Water (100: 16.5: 13.5)

Chloroform: Methanol: 0.26, 0.4, 0.48, 0.27, 0.4 0.58 Water (80 : 20 : 2)

Stem Ethyl acetate: Methanol: 0.17, 0.4, 0.52, 0.39, 0.59 0.59, 0.69, 0.78 Water (100: 16.5: 13.5) Chloroform: Methanol: 0.2, 0.33, 0.44, 0.27, 0.47, 0.6 0.53, 0.66, 0.74 Water (80 : 20 : 2)

Table 13: Antioxidant activity using DPPH assay (% radical scavenging of R. fruticosus )

Conc. %Radical %Radical %Radical %Radical AA %Radical (mg/ml) Scavenging Scavenging Scavenging Scavenging (mg/ml) Scavenging for RFF for RFL for RFR for RFS for AA 5×10 -1 96.00 92.88 90.07 89.88 0.5×10 -1 97.89 5×10 -2 96.00 93.26 91.93 94.27 0.5×10 -2 65.87 5×10 -3 68.97 75.56 74.03 67.06 0.5×10 -3 49.83 5×10 -4 58.47 67.78 68.09 71.26 0.5×10 -4 43.19 5×10 -5 54.17 56.08 63.91 64.10 0.5×10 -5 42.81 5×10 -6 64.82 68.44 64.00 79.28 0.5×10 -6 42.48 5×10 -7 58.94 57.75 59.71 66.01 0.5×10 -7 41.90 5×10 -8 67.06 61.05 61.00 57.37 0.5×10 -8 41.76 5×10 -9 65.63 84.86 68.78 75.84 0.5×10 -9 42.52 5×10 -10 63.72 66.34 62.22 67.35 0.5×10 -10 42.52 AA= Ascorbic acid, Conc. = concentration

Table 14: Antioxidant activity using DPPH assay (% radical scavenging of V. thapsus )

Conc. %Radical %Radical %Radical %Radical AA %Radical (mg/ml) Scavenging Scavenging Scavenging Scavenging (mg/ml) Scavenging for VTF for VTL for VTR for VTS for AA 5×10 -1 89.40 87.73 96.13 85.05 0.5×10 -1 97.89 5×10 -2 73.79 74.30 89.45 80.52 0.5×10 -2 65.87 5×10 -3 58.85 61.19 74.27 66.10 0.5×10 -3 49.83 5×10 -4 63.15 68.30 70.78 66.10 0.5×10 -4 43.19 5×10 -5 56.61 68.30 70.64 66.07 0.5×10 -5 42.81 5×10 -6 60.57 67.54 66.20 64.72 0.5×10 -6 42.48 5×10 -7 62.81 65.91 69.64 61.76 0.5×10 -7 41.90 5×10 -8 58.71 65.68 71.88 53.98 0.5×10 -8 41.76 5×10 -9 56.70 62.62 64.67 58.99 0.5×10 -9 42.52 5×10 -10 55.60 65.58 71.20 57.27 0.5×10 -10 42.52 AA= Ascorbic acid, Conc. = concentration

Table 15: ABTS radical scavenging assay of R. fruticosus and V. thapsus Concentration (% inhibition ± S.E.M) (µg /ml) RFF RFL RFR RFS Ascorbic acid 50 36.19±1.55 24.06±0.65 21.37±1.21 18.77±1.01 69.21±2.01

100 73.02±0.48 50.01±1.07 43.16±0.94 39.15±1.47 96.26±1.91

Concentration (% inhibition ± S.E.M) (µg /ml) VTF VTL VTR VTS Ascorbic acid 50 29.25±1.30 26.50±1.28 21.62±1.35 17.23±1.62 69.21±2.01

100 62.28±0.83 54.42±1.02 45.97±1.62 35.91±1.07 96.26±1.91

Table 16: Nitric oxide radical scavenging assay of R. fruticosus and V. thapsus

Concentration (% inhibition ± S.E.M) (µg/ml) RFF RFL RFR RFS Ascorbic acid 50 23.21±1.03 19.56±0.88 17.75±1.44 13.26±1.31 62.35±2.01

100 47.05±0.69 38.17±1.29 31.07±0.39 26.32±1.66 92.51±1.91

Concentration (% inhibition ± S.E.M) (µg/ml) VTF VTL VTR VTS Ascorbic acid 50 17.08±1.19 14.80±1.52 11.05±1.35 9.61±1.08 62.35±2.01

100 32.99±1.87 26.67±1.23 19.63±1.62 12.32±1.96 92.51±1.91

Table 17: Assessment of (Formalin induced) anti-inflammatory activity

Treatment Dose Mean No. of licking and Inhibition (%) mg/kg biting ±S.E.M orally 1st phase 2nd phase 1st phase 2nd phase Control 0.5 ml 89±3.27 68 ± 3.33 - - Saline R. f fruit 100 mg/kg 73±1.32 65±1.44 17.97 4.41 300 mg/kg 61 ± 1.14 50 ± 0.53 31.46 26.47 500 mg/kg 40 ± 2.34* 30 ± 1.99* 55 55.88 R. f leaves 100 mg/kg 74±0.99 64±1.21 16.85 5.88 300 mg/kg 59+ 1.22 56+ 0.92 33.70 17.64 500 mg/kg 35±1.39* 35±0.73 60.67 48.52 R. f root 100 mg/kg 80±1.23 64±0.83 10.11 5.88 300 mg/kg 78±1.71 60±1.52 12.35 11.76 500 mg/kg 70±1.38 55 ±0.72 21.34 19.11 R. f stem 100 mg/kg 88±1.42 68±1.29 01 0 300 mg/kg 81±0.68 64 +0.89 08 5.8 500 mg/kg 80±0.77 60±0.89 10 11.76 V. t fruit 100 mg/kg 71±3.48 58±1.42 20.22 14.70 300 mg/kg 51±2.93 55±1.54 42.69 19.11 500 mg/kg 39±1.32** 29±2.33 56.17 34.48 V. t leaves 100 mg/kg 70±1.31 63±1.26 21.34 7.35 300 mg/kg 58±1.35 40±0.89 34.83 41.17 500 mg/kg 44±1.24* 35±0.88 50.56 48.52 V. t root 100 mg/kg 78±1.38 61±1.80 12.35 10.29 300 mg/kg 69±1.29 59±1.47 22.47 13.23 500 mg/kg 55±0.53 43±1.29 38.20 36.76 V. t stem 100 mg/kg 81±1.79 71±0.87 08 0 300 mg/kg 79±1.52 65±1.48 11.23 4.41 500 mg/kg 69±0.81 56±0.72 22.47 17.46 Aspirin 300 mg/kg 55± 1.65 48± 1.25 38.20 29.41 Codeine 50 mg/kg 47±4.20 42±4.23 47.19 38.23 sulphate 100 mg/kg 35±1.13* 36±0.53 60.67 47.05 150 mg/kg 23±0.22** 33±0.40* 74.15 51.47 Mean + S.E.M; N = 5; Significance with respect to control (* = Significant results, ** = highly significant results)

Table 18: Assessment of (Carrageenan induced) anti-inflammatory activity

Treatment Dose Mean diameter of rat paw in mm ± % of inhibition mg/kg S.E.M orally 1hr 2hr 3hr 1hr 2hr 3hr Control 0.5 ml 16.8 ±1.67 20 ± 2.37 24.5 ±1.97 - - - Saline R. f fruit 100 mg/kg 11.8±1.32 12.1±1.31 20.3 ±1.12 29.76 39.5 17.14 300 mg/kg 11.6 ±1.29 11.9 ± 2.23* 18.7 ±1.13 30.95 40.5 23.67 500 mg/kg 11.1±1.24* 11.5 ± 0.81 17.2 ±1.89 33.92 42.5 29.79 R. f leaves 100 mg/kg 12.2±1.49 14.5 ±1.15 19.3±1.25 27.38 27.5 21.22 300 mg/kg 12± 1.14 12.7 ±1.72 18.3±1.23* 28.57 36.5 25.30 500 mg/kg 10.5±0.83** 11.4±1.42** 15.3±0.73* 37.54 43 37.55 R. f root 100 mg/kg 16.1±1.13 17.3 ±1.43 22.8 ±1.73 4.16 13.5 6.93 300 mg/kg 15.4±1.71 16.8 ±1.42 20.9±0.43* 8.33 16 14.69 500 mg/kg 14.3 ±1.22 16.2±1.28 19.6±1.52 14.88 19 20 R. f stem 100 mg/kg 15.3±1.42 16.8 ±1.32 23.2 ±1.31 8.92 16 5.30 300 mg/kg 14.2 ±1.49 16.4 ±1.38 22.1 ±1.99 15.47 18 9.79 500 mg/kg 14 ±1.79 14.3±1.47 19.9 ±1.89 16.66 28.5 18.77 V. t fruit 100 mg/kg 12.6±2.48 13.1±1.52 21.9±1.42 25 34.5 10.61 300 mg/kg 12.2±1.93 12.9±1.34 20.1±1.44 27.38 35.5 17.95 500 mg/kg 11.5±2.33 12.2±1.52* 18±1.44 31.54 39 26.53 V. t leaves 100 mg/kg 12.3±1.41 12.8 ± 1.36 21.3±1.42 26.78 36 13.06 300 mg/kg 12.2±1.29 12.5±1.36* 20.3±1.35 27.38 37.5 17.14 500 mg/kg 11.2 ±1.98* 12.7±1.44* 18.7±1.42 33.33 36.5 23.67 V. t root 100 mg/kg 14.5±1.40 14.9±1.40 21.3±1.44 13.69 25.5 13.06 300 mg/kg 14±1.27 14.5±1.29 21.1±1.24 16.66 27.5 13.87 500 mg/kg 13.8±1.29 13.7±0.93 20±1.74 17.85 31.5 18.36 V. t stem 100 mg/kg 15±1.17 15.7±0.90 22.3 ±1.52 10.71 21.5 8.97 300 mg/kg 14.3±1.28 14.9±1.62 21.3±1.42 14.88 25.5 13.06 500 mg/kg 13.7±0.92 13.8±1.82 19.5±1.52 18.45 31 20.40 Aspirin 300 mg/kg 12.5± 1.34* 15.2± 0.84 16.3±0.72* 25.59 24.0 33.46 Mean + S.E.M; N = 5; Significance with respect to control. * = Significant results, ** = highly significant results.

Table 19: Effect of crude extract of on Hot plate Analgesiometer in mice

Variation in flicking time with ± SEM

(Time in sec at 55 ± 1ºC)

Group 0hr 0.5hr 1hr 1.5hrs 2hrs 2.5hrs 3hrs 3.5hrs 4hrs 4.5hrs Control 11.2± 11.4± 11.4± 10.4± 12.2± 12.2± 11.9± 12.3± 10.6± 11.2± 1.19 1.14 1.83 1.49 1.72 1.16 1.12 1.11 1.21 1.02 R. f fruit 11.2± 15.2± 17.3± 20.3± 22.4± 25.3± 25.3± 22± 20.5± 17± 100 mg/kg 0.95 0.86 1.05 0.82 0.62 0.79 0.59 1.24 2.11 1.13 R. f fruit 12.8± 24± 28.3± 26.8± 33.2*± 35.4± 25± 20.6± 18.8± 15.6± 300 mg/kg 0.92 1.24 1.25 1.33 1.05 0.89 1.29 1.54 2.18 1.43 R. f fruit 14.2± 27.6± 24± 27.2± 34.4*± 29.6± 27.8± 26.2± 19.2± 16± 500 mg/kg 1.79 0.70 1.19 1.66 1.69 1.39 1.12 1.03 1.83 2.18 R. f leaves 12.5± 13.7 ± 16.2 ± 16.3± 16.2± 16.3± 14.4± 14.2± 14.1± 13± 100 mg/kg 0.76 1.17 1.18 1.16 1.70 1.71 1.17 1.99 1.83 1.28 R. f leave s 13.1± 13.9± 17.7± 20.2± 22.2± 16.8± 16.2± 12.2± 12.2± 11.8± 300 mg/kg 0.71 0.82 0.86 1.17 0.80 1.59 0.86 2.13 2.13 1.28 R. f leaves 10.4± 18± 25.4± 28± 35.8*± 24.8± 22.8± 18.2± 15.4± 14.4± 500 mg/kg 0.90 0.71 0.92 0.79 0.82 1.70 0.62 2.07 1.17 1.24 R. f root 10.6± 13 ± 15.2 ± 17.3± 19.2 ± 20.5± 18.3± 16.2 ± 14.2± 12.2± 100 mg/kg 0.92 0.71 1.12 0.89 1.19 0.96 1.14 1.24 1.05 1.55 R. f root 11.7± 15.2 ± 18 ± 22± 25.3± 25.8 ± 22.3± 20.5 ± 15.6± 13.3± 300 mg/kg 1.12 1.09 1.16 0.49 0.82 1.67 0.79 1.76 1.43 1.82 R. f root 10.6± 13 ± 15.2 ± 17.3± 19.2 ± 20.5± 18.3± 16.2 ± 14.2± 12.2± 500 mg/kg 0.92 0.71 1.12 0.89 1.19 0.96 1.14 1.24 1.05 1.55 R. f stem 10.2± 12.5± 13± 13.5± 14± 14.5± 14.3± 14.3± 13.8± 12.6± 100 mg/kg 1.07 0.92 1.07 0.70 0.80 1.24 1.93 1.02 1.28 1.13 R. f stem 11± 13± 15± 15.5± 17± 18± 17.4± 16± 14.2 12.5± 300 mg/kg 0.79 0.60 1.70 0.70 0.99 1.02 0.72 0.84 ±0.61 1.11 R. f stem 9.4± 10.5± 17± 17± 18.2± 15.8± 13.8± 13.8± 12.1± 11.7± 500 mg/kg 0.29 0.79 0.59 1.02 1.84 1.17 1.90 1.19 1.41 1.02 Aspirin 13 ± 36* ± 36* ± 40* ± 42* ± 46**± 35 ± 24 ± 24± 20± 300 mg/kg 1.23 0.26 0.72 0.32 1.31 1.16 0.71 0.61 0.11 0.81 Values represent the mean SEM. Statistically significant from control and standard drug. * Significant, ** Highly significant

Table 20: Effect of crude extract on Hot plate Analgesiometer in mice

Variation in flicking time with ± SEM

(Time in sec at 55 ± 1ºC)

Group 0hr 0.5hr 1hr 1.5hrs 2hrs 2.5hrs 3hrs 3.5hrs 4hrs 4.5hrs Control 11.2± 11.4± 11.4± 10.4± 12.2± 12.2± 11.9± 12.3± 10.6± 11.2± 1.19 1.14 1.83 1.49 1.72 1.16 1.12 1.11 1.21 1.02 V. t fruit 12.4± 17.3± 18.4± 19.3± 23± 22.3± 17.3± 15.3± 13.2± 12.2± 100 mg/kg 0.78 0.45 1.42 0.82 0.89 1.69 1.19 1.14 2.11 1.13 V. t fruit 13.2± 19.2± 22.4± 25.3± 28.2± 28.6± 20.8± 19.3± 15.4± 14.2± 300 mg/kg 0.82 1.28 1.09 1.33 1.72 0.88 0.78 1.02 2.18 1.13 V. t fruit 14.1± 21.2± 25.3± 29.3± 35*± 30.4± 25.4± 18.4± 16.5± 14.3± 500 mg/kg 0.92 0.70 0.92 1.10 0.99 0.97 0.81 2.01 1.03 2.10 V. t leaves 12.2± 16.3 ± 19.3 ± 24.4± 27.3± 30.4± 28.3± 22.3± 18.3± 12.8± 100 mg/kg 0.62 1.07 1.28 1.46 1.70 1.79 1.17 1.03 1.11 2.18 V. t leaves 13± 18.3± 17.6± 26.4± 35.2*± 33.4± 30.3± 21.4± 19.5± 13.5± 300 mg/kg 1.09 0.82 0.87 1.14 1.12 1.59 1.12 1.03 2.13 2.18 V. t leaves 14.3± 19.6 22.3± 29± 37.8*± 35.4*± 31± 26.5± 21.4± 15.3± 500 mg/kg 0.93 0.89 1.92 1.39 1.72 1.20 0.80 2.07 1.07 1.14 V. t root 10.2± 16.3 ± 18.4 ± 19± 20.4 ± 18.3± 16.2± 14.4 ± 13.2± 11.3± 100 mg/kg 0.62 0.70 0.86 0.75 0.93 0.97 1.17 1.20 1.33 1.50 V. t root 12.2± 17.5 ± 19.3 ± 21.6± 21.8± 25 ± 22.3± 15.4 ± 13.4± 12.5± 300 mg/kg 0.80 0.89 1.16 0.90 0.62 1.36 0.70 1.76 1.53 1.80 V. t root 13.4± 19.5± 22.3± 24.4± 24.2± 28.2± 19.8± 17.4± 15.4± 14.1± 500 mg/kg 1.22 1.03 1.21 1.20 0.58 0.82 1.07 1.40 1.29 1.22 V. t stem 11.2± 15.3± 17.3± 19± 20± 19.8± 17.4± 17.4± 13.2± 12.4± 100 mg/kg 1.19 1.69 1.27 0.85 0.87 1.24 1.36 1.04 1.28 1.13 V. t stem 11.8± 16.4± 18.4± 18.9± 20.6± 20.4± 16.4± 17.6± 15.6 13.2± 300 mg/kg 0.79 0.68 1.17 1.70 0.90 1.32 0.74 0.84 ±0.73 1.17 V. t stem 10.4± 17.3± 18.3± 22± 23± 22.5± 18.4± 16.2± 14.3± 14.4± 500 mg/kg 0.97 1.19 1.71 1.13 1.40 1.15 1.90 1.41 1.45 1.20 Aspirin 13 ± 36* ± 36* ± 40* ± 42* ± 46**± 35 ± 24 ± 24± 20± 300 mg/kg 1.23 0.26 0.72 0.32 1.31 1.16 0.71 0.61 0.11 0.81 Values represent the mean SEM. Statistically significant from control and standard drug. * Significant, ** Highly significant

Table 21: Assessment of analgesic activity (Acetic acid induced writhing)

Treatment Dose Mean No. of Writhes + Inhibition (%) mg/kg S.E.M orally 1st phase 2nd phase 1st phase 2nd phase Control 0.5 ml 75+ 4.36 49 + 2.81 - - Saline R. f fruit 100 mg/kg 49.67±0.90 39.7±2.21 33.77 18.97 300 mg/kg 37.5+ 1.22 30.39+ 0.92 50.00 37.97 500 mg/kg 29.2±1.72* 18±0.73* 61.06 63.26 R. f leaves 100 mg/kg 41±1.32 29.2±1.24 45.33 40.40 300 mg/kg 28.6 + 1.394 24.7 + 0.43 61.86 49.59 500 mg/kg 15.2 + 1.34** 11 + 0.89** 79.73 77.55

R. f root 100 mg/kg 42.4 ±1.23 32.7±0.93 43.46 33.26 300 mg/kg 31.2 ±1.71* 22.6±1.62* 58.4 53.87 500 mg/kg 29.3±1.38* 25.2±1.52 60.93 48.57 R. f stem 100 mg/kg 70±1.21 49±1.49 06.66 0 300 mg/kg 61.3±0.48 48.2 +0.79 18.26 1.63 500 mg/kg 58.6±0.77 39.21±0.69 21.86 19.97 V. t fruit 100 mg/kg 39.3±3.48 32.1±1.32 47.60 34.48 300 mg/kg 30.1±1.93 21.2±1.34 59.86 56.73 500 mg/kg 19.7±1.18** 18.2±2.33* 73.73 62.85 V. t leaves 100 mg/kg 39.47±1.31 30.1±1.36 47.37 38.57 300 mg/kg 32.8±1.60 22.2±0.79 56.26 54.69 500 mg/kg 27±2.14* 20.3±0.88* 64.00 58.57 V. t root 100 mg/kg 59.2±1.38 41.7±1.80 21.06 14.89 300 mg/kg 52.3±1.29 39.1±1.37 30.26 20.20 500 mg/kg 47.3±0.83 25.5±1.29 36.93 47.95 V. t stem 100 mg/kg 69.9±1.99 48.3±0.87 6.80 01.42 300 mg/kg 60±2.32 41±1.38 20.00 16.32 500 mg/kg 49.9±0.97 31.3±1.82 33.46 36.12 Aspirin 300 mg/kg 44.4+ 1.05 20 + 0.45* 40.80 59.18 Mean + S.E.M; N = 5; Significance with respect to control (* = Significant results, ** = highly significant results)

Table 22: Effect of crude extract on water bath (tail flick) in mice

Variation in flicking time with ± SEM

(Time in sec at 51 ± 1ºC)

Group 0hr 0.5hr 1hr 1.5hrs 2hrs 2.5hrs 3hrs 3.5hrs 4hrs 4.5hrs min Control 1.6 1.80 1.80 1.07 1.6 1.30 1.20 2.0 1.4 1.2 ±0.19 ±0.17 ±0.08 ±0.17 ±0.11 ±0.11 ±0.16 ±0.08 ±0.11 ±0.08 R. f fruit 1.65 2.3 2.99 3.2 3.4 3.4 2.8 2.8 2.6 2.2 100 mg/kg ±0.11 ±0.22 ±0.19 ±0.24 ±0.25 ±0.15 ±0.18 ±0.28 ±0.22 ±0.23 R. f fruit 1.7 3.2 3.8 4.2 4.7* 5.2* 4.3 4.1 3.8 3.2 300 mg/kg ±0.20 ±0.27 ±0.33 ±0.35 ±0.15 ±0.33 ±0.11 ±0.21 ±0.16 ±0.19 R. f fruit 2.4 4.2* 4.8* 5.67* 6.22* 6.8** 5.3* 5.4* 4.4* 3.8 500 mg/kg ±0.17 ±0.28 ±0.22 ±0.11 ±0.19 ±0.13 ±0.23 ±0.33 ±0.25 ±0.29 R. f leaves 1.5 1.8 1.9 1.40 1.04 1.30 1.20 1.5 1.40 1.40 100 mg/kg ±0.11 ±0.17 ±0.23 ±0.15 ±0.13 ±0.21 ±0.17 ±0.11 ±0.12 ±0.10 R. f leaves 1.6 3.7* 3.7* 3.8* 4.2* 4.4* 3.2 3.2 2.1 2.3 300 mg/kg ±0.23 ±0.13 ±0.17 ±0.12 ±0.23 ±0.31 ±0.22 ±0.24 ±0.23 ±0.25 R. f leaves 1.4 4.2 4.7* 5.2* 6.28** 6.4* 6.00* 5.00* 4.1 3.2 500 mg/kg ±0.12 ±0.33 ±0.22 ±0.23 ±0.19 ±0.20 ±0.11 ±0.10 ±0.30 ±0.15 R. f root 1.2 2.2 2.9 2.8 2.7 3.00 2.70 2.7 2.2 1.8 100 mg/kg ±0.33 ±0.10 ±0.12 ±0.21 ±0.20 ±0.19 ±0.13 ±0.31 ±0.17 ±0.10 R. f root 1.37 2.4 2.4 2.9 3.00 3.2 3.3 2.9 2.7 2.00 300 mg/kg ±0.19 ±0.25 ±0.20 ±0.31 ±0.32 ±0.23 ±0.24 ±0.13 ±0.29 ±0.18 R. f root 1.40 3.2 3.3 3.7* 4.2* 4.2* 3.3 3.7 2.5 2.5 500 mg/kg ±0.26 ±0.21 ±0.20 ±0.31 ±0.39 ±0.15 ±0.24 ±0.13 ±0.22 ±0.13 R. f stem 1.34 1.60 2.2 2.4 2.4 2.00 1.8 1.70 1.6 1.4 100 mg/kg ±0.15 ±0.14 ±0.16 ±0.15 ±0.23 ±0.12 ±0.10 ±0.14 ±0.19 ±0.23 R. f stem 1.56 1.9 1.95 2.7 2.7 2.9 2.3 2.00 1.8 1.6 300 mg/kg ±0.13 ±0.18 ±0.26 ±0.20 ±0.10 ±0.26 ±0.21 ±0.13 ±0.35 ±0.16 R. f stem 1.6 1.8 2.1 2.4 2.9 3.00 2.2 2.2 2.4 1.9 500 mg/kg ±0.12 ±0.14 ±0.17 ±0.14 ±0.19 ±0.21 ±0.20 ±0.16 ±0.14 ±0.22 Aspirin 1.20 4.4* 3.5* 3.8* 5.9* 5.0** 4.0* 3.2 2.8 2.5 300 mg/kg ±0.15 ±1.04 ±0.34 ±0.22 ±0.35 ±0.33 ±0.21 ±1.03 ±0.25 ±0.16 Mean + S.E.M; N = 5; Significance with respect to control. * = Significant results, ** = highly significant results .

Table 23: Effect of crude extract on water bath (tail flick) in mice

Variation in flicking time with ± SEM

(Time in sec at 51 ± 1ºC)

Group 0hr 0.5hr 1hr 1.5hrs 2hrs 2.5hrs 3hrs 3.5hrs 4hrs 4.5hrs min Control 1.6 1.80 1.80 1.07 1.6 1.30 1.20 2.0 1.4 1.2 ±0.19 ±0.17 ±0.08 ±0.17 ±0.11 ±0.11 ±0.16 ±0.08 ±0.11 ±0.08 V. t fruit 2.4 3.6* 3.9* 3.8* 3.4* 3.2 3.2 2.7 2.4 2.4 100 mg/kg ±0.10 ±0.12 ±0.19 ±0.34 ±0.25 ±0.21 ±0.19 ±0.11 ±0.21 ±0.23 V. t fruit 1.8 4.7* 5* 5.2* 4.8* 4.6* 4.4* 3.8* 3.8* 3.00 300 mg/kg ±0.20 ±0.29 ±0.33 ±0.31 ±0.25 ±0.13 ±0.35 ±0.20 ±0.16 ±0.19 V. t fruit 1.4 5.2* 5.6* 6.2* 6.4** 6.00* 5.3* 5.2* 4.2* 3.8 500 mg/kg ±0.17 ±0.28 ±0.26 ±0.31 ±0.35 ±0.13 ±0.23 ±0.23 ±0.45 ±0.19 V. t leaves 1.5 2.9 3.7* 3.8* 4.2* 4.2* 3.2 3.4 2.9 2.8 100 mg/kg ±0.22 ±0.26 ±0.31 ±0.25 ±0.13 ±0.23 ±0.29 ±0.17 ±0.16 ±0.13 V. t leaves 2.3 4.5* 4.8* 5.4* 5.9* 4.4* 3.9* 2.8 2.8 2.2 300 mg/kg ±0.12 ±0.14 ±0.11 ±0.22 ±0.33 ±0.33 ±0.20 ±0.17 ±0.23 ±0.32 V. t leaves 2.00 5.2* 5.8* 6.2** 6.5** 6.2** 5.8* 4.8* 3.4 2.8 500 mg/kg ±0.17 ±0.33 ±0.37 ±0.36 ±0.19 ±0.32 ±0.37 ±0.12 ±0.13 ±0.09 V. t root 1.4 2.3 2.9 3.2 3.3 3.4 2.70 2.8 2.8 2.4 100 mg/kg ±0.23 ±0.26 ±0.32 ±0.26 ±0.14 ±0.26 ±0.17 ±0.33 ±0.23 ±0.12 V. t root 1.35 3.0 3.4 3.9* 3.8* 3.4 3.00 2.8 2.8 2.00 300 mg/kg ±0.11 ±0.25 ±0.21 ±0.21 ±0.32 ±0.33 ±0.21 ±0.23 ±0.19 ±0.19 V. t root 1.7 3.96* 4.2* 4.6* 4.7* 4.2* 3.8* 3.3 3.2 3.00 500 mg/kg ±0.35 ±0.22 ±0.15 ±0.31 ±0.39 ±0.35 ±0.34 ±0.23 ±0.32 ±0.10 V. t stem 1.2 1.9 2.5 2.8 3.2 3.2 2.8 2.8 1.8 1.9 100 mg/kg ±0.07 ±0.18 ±0.27 ±0.16 ±0.30 ±0.11 ±0.23 ±0.25 ±0.13 ±0.31 V. t stem 1.70 2.6 3.2 3.2 3.8* 3.4 3.2 3.4 2.9 2.2 300 mg/kg ±0.12 ±0.17 ±0.26 ±0.33 ±0.25 ±0.10 ±0.21 ±0.16 ±0.35 ±0.27 V. t stem 2.1 3.9* 3.7 3.2 3.7 3.2 2.9 2.8 2.7 2.7 500 mg/kg ±0.13 ±0.34 ±0.24 ±0.14 ±0.33 ±0.32 ±0.21 ±0.17 ±0.24 ±0.22 Aspirin 1.20 4.4 3.5* 3.8* 5.9* 5.0** 4.0* 3.2 2.8 2.5 300 mg/kg ±0.15 ±1.04* ±0.34 ±0.22 ±0.35 ±0.33 ±0.21 ±1.03 ±0.25 ±0.16 Mean + S.E.M; N = 5; Significance with respect to control. * = Significant results, ** = highly significant results .

Table 24: Diuretic activity of R. fruticosus and V. thapsus extracts

Treatment Dose Volume of urine(ml) Diuretic Index within 3 hours ±SEM Control - 0.06±0.187 - R. f fruit 100 mg/kg 0.36±0.087** 6 300 mg/kg 0.44±0.067** 7.33 500 mg/kg 0.296±0.056* 4.93 R. f leaves 100 mg/kg 0.03±0.004 0.5 300 mg/kg 0.28±0.066* 4.66 500 mg/kg 0.04±0.002 0.66 R. f root 100 mg/kg 0.02±0.002 0.33 300 mg/kg 0.02±0.003 0.33 500 mg/kg 0.062±0.009 1.03 R. f stem 100 mg/kg 0.15±0.027* 2.5 300 mg/kg 0.47±0.104** 7.83 500 mg/kg 0.27±0.094 4.5 V. t fruit 100 mg/kg 0.22±0.034** 3.66 300 mg/kg 0.25±0.005 4.16 500 mg/kg 0.04±0.021 0.66 V. t leaves 100 mg/kg 0.06±0.040 1 300 mg/kg 0.02±0.020 0.33 500 mg/kg 0.04±0.024 0.66 V. t root 100 mg/kg 0.05±0.015 0.83 300 mg/kg 0.05±0.022 0.83 500 mg/kg 0.06±0.018 1 V. t stem 100 mg/kg 0.08±0.021 1.33 300 mg/kg 0.03±0.009 0.5 500 mg/kg 0.12±0.021 2 Hydrochlorothiazide 10 mg/kg 0.51±0.10** 8.5 Statistically significant* Very significant**

Table 25: Behavioral responses of R. fruticosus fruit in mice

PARAMETERS R. fruticosus fruit 100 mg/kg Control 0 min 5 min. 10 15 30 60min 2 hrs 4 hrs 24hrs min min min Lacrimation ------Pupil size ------Nystagmus ------Salivation ------Vocalization ------Pilo erection ------Micturition - - - - - + + + ++ - Irritability ------Disorientation /Staggering gate ------Head tap. aggressive ------Passivity ------Spontaneous activity + + + + ------Dec. motor activity - - - - - + + + - - Dec. resp. rate ------Dec. resp. depth ------Increase resp. rate - - - + + + + - - - Increase resp. depth ------Dyspnea ------Analgesia/pain response - - - ↓+ ↓+ ↓+ ↓+ - - - Anesthesia ------Corneal reflex ------Light reflex ------Ataxia ------Limb tone decrease ------Body tone - - - + + + + + + - Tail erection ------Tail lashing ------Enophthlmoses ------Exophthalmoses ------Touch response - - - + + + + + + - Tremor ------Straub tail ------Righting reflex ------

Table 26: Behavioral responses of R. fruticosus fruit in mice

PARAMETERS R. fruticosus fruit 300 mg/kg Control 0 min 5 min. 10 15 30 60min 2 hrs 4 hrs 24hrs min min min Lacrimation ------Pupil size ------Nystagmus ------Salivation ------Vocalization ------Pilo erection ------Micturition - - - - - + + ++ + - Irritability ------Disorientation /Staggering gate ------Head tap. aggressive ------Passivity - - ↓+ ↓+ ↓+ ↓+ - - - - Spontaneous activity + + + + ------Dec. motor activity ------Dec. resp. rate ------Dec. resp. depth ------Increase resp. rate - - + + + + + + + - Increase resp. depth ------Dyspnea ------Analgesia/pain response - - - ↓+ ↓+ ↓+ ↓+ - - - Anesthesia ------Corneal reflex ------Light reflex ------Ataxia ------Limb tone decrease ------Body tone - - - + + + + + + - Tail erection ------Tail lashing ------Enophthlmoses ------Exophthalmoses ------Touch response - - - + + + + + + - Tremor ------Straub tail ------Righting reflex ------

Table 27: Behavioural responses of R. fruticosus fruit in mice

PARAMETERS R. fruticosus fruit 500 mg/kg Control 0 5 min. 10 15 30 60min 2 hrs 4 hrs 24hrs min min min min Lacrimation ------Pupil size ------Nystagmus ------Salivation ------Vocalization ------Pilo erection ------Micturition ------+ + + - Irritability ------Disorientation /Staggering gate ------Head tap. aggressive ------Passivity - - ↓+ ↓+ ↓+ - - - - - Spontaneous activity + + + + ------Dec. motor activity ------Dec. resp. rate ------Dec. resp. depth ------Increase resp. rate - - + + + + + + + - Increase resp. depth - - - - - + + + + - Dyspnea ------Analgesia/pain response - - - ↓+ ↓+ ↓+ ↓+ ↓ - - Anesthesia ------Corneal reflex ------Light reflex ------Ataxia ------Limb tone decrease ------Body tone - - + + + + + + + - Tail erection ------Tail lashing ------Enophthlmoses ------Exophthalmoses ------Touch response - - + + + + + + + - Tremor ------Straub tail ------Righting reflex ------

Table 28: Behavioural response of R. fruticosus leaves in mice

PARAMETERS R. fruticosus leaves 100 mg/kg Control 0 min 5 min. 10 min 15 min 30 min 60min 2 hrs 4 hrs 24hrs Lacrimation ------Pupil size ------Nystagmus ------Salivation ------Vocalization ------Pilo erection ------Micturition ------+ + - - Irritability - - + + ------Disorientation /Staggering gate ------Head tap. aggressive ------Passivity - - ↓+ ↓+ ↓+ - - - - - Spontaneous activity + + + + ------Dec. motor activity ------Dec. resp. rate ------Dec. resp. depth ------Increase resp. rate - - + + + + + - - - Increase resp. depth - - + + + + + - - - Dyspnea ------Analgesia/pain response - - - - - ↓+ ↓+ - - - Anesthesia ------Corneal reflex ------Light reflex ------Ataxia ------Limb tone decrease ------Body tone - - - + + + + + + - Tail erection ------Tail lashing ------Enophthlmoses ------Exophthalmoses ------Touch response - - + + + + + + - - Tremor ------Straub tail ------Righting reflex ------

Table 29: Behavioral responses of R. fruticosus leaves in mice

PARAMETERS R. fruticosus leaves 300 mg/kg Control 0 min 5 min. 10 15 30 60min 2 hrs 4 hrs 24hrs min min min Lacrimation ------Pupil size ------Nystagmus ------Salivation ------Vocalization ------Pilo erection ------Micturition ------+ + - Irritability ------Disorientation /Staggering gate ------Head tap. aggressive ------Passivity - - ↓+ ↓+ ↓+ - - - - - Spontaneous activity + + + + ------Dec. motor activity ------Dec. resp. rate ------Dec. resp. depth ------Increase resp. rate - - + + + + + + - - Increase resp. depth - - + + + + + + - - Dyspnea ------Analgesia/pain response - - - ↓+ ↓+ ↓+ ↓+ - - - Anesthesia ------Corneal reflex ------Light reflex ------Ataxia ------Limb tone decrease ------Body tone - - + + + + + + - - Tail erection ------Tail lashing ------Enophthlmoses ------Exophthalmoses ------Touch response - - + + + + + + - - Tremor ------Straub tail ------Righting reflex ------

Table 30: Behavioral responses of R. fruticosus leaves in mice

PARAMETERS R. fruticosus leaves 500 mg/kg Control 0 min 5 min. 10 15 30 60min 2 hrs 4 hrs 24hrs min min min Lacrimation ------Pupil size ------Nystagmus ------Salivation ------Vocalization ------Pilo erection ------Micturition ------+ + + - Irritability ------Disorientation /Staggering gate ------Head tap. aggressive ------Passivity - - ↓+ ↓+ ↓+ - - - - - Spontaneous activity + + + + ------Dec. motor activity ------Dec. resp. rate ------Dec. resp. depth ------Increase resp. rate - - - ++ ++ ++ ++ + + - Increase resp. depth - - - ++ ++ ++ + + + - Dyspnea ------Analgesia/pain response - - - - ↓+ ↓+ ↓+ ↓+ - - Anesthesia ------Corneal reflex ------Light reflex ------Ataxia ------Limb tone decrease ------Body tone - - + + + + + + + - Tail erection ------Tail lashing ------Enophthlmoses ------Exophthalmoses ------Touch response - - + + + + + + + - Tremor ------Straub tail ------Righting reflex ------

Table 31: Behavioral response of R. fruticosus root in mice

PARAMETERS R. fruticosus root 100 mg/kg Control 0 min 5 min. 10 15 30 60min 2 hrs 4 hrs 24hrs min min min Lacrimation ------Pupil size ------Nystagmus ------Salivation ------Vocalization ------Pilo erection ------Micturition ------Irritability ------Disorientation /Staggering gate ------Head tap. aggressive ------Passivity - - - ↓+ ↓+ - - - - - Spontaneous activity - + + + - - - - Dec. motor activity ------Dec. resp. rate ------Dec. resp. depth ------Increase resp. rate - - - - + + + + - - Increase resp. depth ------Dyspnea ------Analgesia/pain response ------↓+ - - - Anesthesia ------Corneal reflex ------Light reflex ------Ataxia ------Limb tone decrease ------Body tone - - - - + + + + - - Tail erection ------Tail lashing ------Enophthlmoses ------Exophthalmoses ------Touch response - - - - + + + + - - Tremor ------Straub tail ------Righting reflex ------

Table 32: Behavioral response of R. fruticosus root in mice

PARAMETERS R. fruticosus root 300 mg/kg Control 0 min 5 min. 10 15 30 60min 2 hrs 4 hrs 24hrs min min min Lacrimation ------Pupil size ------Nystagmus ------Salivation ------Vocalization ------Pilo erection ------Micturition ------Irritability ------Disorientation /Staggering gate ------Head tap. aggressive ------Passivity - - - - ↓+ ↓+ ↓+ - - - Spontaneous activity + + + + + - - - Dec. motor activity ------Dec. resp. rate ------Dec. resp. depth ------Increase resp. rate - - + + + + + + - - Increase resp. depth - - - + + + + + - - Dyspnea ------Analgesia/pain response - - - ↓+ ↓+ ↓+ ↓+ - - - Anesthesia ------Corneal reflex ------Light reflex - - - + + + + + - - Ataxia ------Limb tone decrease ------Body tone - + + + + + + + + - Tail erection ------Tail lashing ------Enophthlmoses ------Exophthalmoses ------Touch response - - - + + + + + + - Tremor ------Straub tail ------Righting reflex ------

100

Table 33: Behavioral responses of R. fruticosus root in mice

PARAMETERS R. fruticosus root 500 mg/kg Control 0 min 5 min. 10 15 30 60min 2 hrs 4 hrs 24hrs min min min Lacrimation ------Pupil size ------Nystagmus ------Salivation ------Vocalization ------Pilo erection ------Micturition ------Irritability ------Disorientation /Staggering gate ------Head tap. aggressive ------Passivity - - - - ↓+ ↓+ ↓+ - - - Spontaneous activity - - - + + + + + - - Dec. motor activity ------Dec. resp. rate ------Dec. resp. depth ------Increase resp. rate - - - + + + + + + - Increase resp. depth - - - + + + + + + - Dyspnea ------Analgesia/pain response - - - ↓+ ↓+ ↓+ ↓+ ↓+ - - Anesthesia - - - - + + - - - - Corneal reflex ------Light reflex - - - - - + + + + - Ataxia ------Limb tone decrease ------Body tone - - - + + ++ + + + - Tail erection ------Tail lashing ------Enophthlmoses ------Exophthalmoses ------Touch response - - - + + + ++ + + - Tremor ------Straub tail ------Righting reflex ------

101

Table 34: Behavioural response of R. fruticosus stem in mice

PARAMETERS R. fruticosus stem 100 mg/kg Control 0 min 5 min. 10 15 30 60min 2 hrs 4 hrs 24hrs min min min Lacrimation ------Pupil size ------Nystagmus ------Salivation ------Vocalization ------Pilo erection ------Micturition ------+ + + - Irritability - - - - + + - - - - Disorientation /Staggering gate ------Head tap. aggressive ------Passivity - - - ↓+ ↓+ ↓+ - - - - Spontaneous activity - + + + ------Dec. motor activity ------Dec. resp. rate ------Dec. resp. depth ------Increase resp. rate - - - - + + + + - - Increase resp. depth ------+ + - - Dyspnea ------Analgesia/pain response - - - - - ↓+ ↓+ - - - Anesthesia ------Corneal reflex ------Light reflex ------Ataxia ------Limb tone decrease ------Body tone - - - + + + + + + - Tail erection ------Tail lashing ------Enophthlmoses - - + + + - - - - - Exophthalmoses - - - - - + + - - - Touch response - - - + + + + + + - Tremor ------Straub tail ------Righting reflex ------

102

Table 35: Behavioural response of R. fruticosus stem in mice

PARAMETERS R. fruticosus stem 300 mg/kg Control 0 min 5 min. 10 min 15 min 30 min 60min 2 hrs 4 hrs 24hrs Lacrimation ------Pupil size ------Nystagmus ------Salivation ------Vocalization ------Pilo erection ------Micturition ------+ + ++ - Irritability ------Disorientation /Staggering gate ------Head tap. aggressive ------Passivity - - - ↓+ ↓+ ↓+ - - - - Spontaneous activity + + + ++ ++ -- - - Dec. motor activity ------Dec. resp. rate ------Dec. resp. depth ------Increase resp. rate - - - - + + + + - - Increase resp. depth - - - - + + + + - - Dyspnea ------Analgesia/pain response - - - - ↓+ ↓+ ↓+ ↓+ - - Anesthesia ------Corneal reflex ------Light reflex ------Ataxia ------Limb tone decrease ------Body tone - - - - + + + ++ + - Tail erection ------Tail lashing ------Enophthlmoses ------Exophthalmoses ------Touch response - - - - + + + + ++ - Tremor ------Straub tail ------Righting reflex ------

103

Table 36: Behavioral response of R. fruticosus stem in mice

PARAMETERS R. fruticosus stem 500 mg/kg Control 0 min 5 min. 10 15 30 60min 2 hrs 4 hrs 24hrs min min min Lacrimation ------Pupil size ------Nystagmus ------Salivation ------Vocalization ------Pilo erection ------Micturition ------+ + - - Irritability ------Disorientation /Staggering gate ------Head tap. aggressive ------Passivity - - - ↓+ ↓+ ↓+ ↓+ - - - Spontaneous activity - + + + ++ + - - Dec. motor activity ------Dec. resp. rate ------Dec. resp. depth ------Increase resp. rate - - - - ++ ++ ++ + + - Increase resp. depth - - - - ++ ++ + + + - Dyspnea ------Analgesia/pain response - - - - ↓+ ↓+ ↓+ + - - - Anesthesia - - - - + + - - - - Corneal reflex ------Light reflex ------Ataxia ------Limb tone decrease ------Body tone - - - + + + + + ++ - Tail erection ------Tail lashing ------Enophthlmoses ------Exophthalmoses ------Touch response - - - + + + + ++ - - Tremor ------Straub tail ------Righting reflex ------

104

Table 37: Behavioural responses of V. thapsus fruit in mice

PARAMETERS V. thapsus fruit 100 mg/kg Control 0 min 5 min. 10 15 30 60min 2 hrs 4 hrs 24hrs min min min Lacrimation ------Pupil size ------Nystagmus ------Salivation ------Vocalization ------Pilo erection ------Micturition ------+ + + - Irritability ------Disorientation /Staggering gate ------Head tap. aggressive ------Passivity - - + + + - - - - - Spontaneous activity - - ↓+ ↓+ ------Dec. motor activity - - - - - ↓+ ↓+ ↓+ - - Dec. resp. rate ------Dec. resp. depth ------Increase resp. rate ------Increase resp. depth ------Dyspnea ------Analgesia/pain response - - - - - ↓+ ↓+ - - - Anesthesia ------Corneal reflex ------Light reflex ------Ataxia ------Limb tone decrease ------Body tone - - + + + + + - - - Tail erection - - + + + + - - - - Tail lashing ------Enophthlmoses ------Exophthalmoses ------Touch response - - - - - ↓+ ↓+ - - - Tremor ------Straub tail ------Righting reflex ------

105

Table 38: Behavioral responses of V. thapsus fruit in mice

PARAMETERS V. thapsus fruit 300 mg/kg Control 0 min 5 min. 10 15 30 60min 2 hrs 4 hrs 24hrs min min min Lacrimation ------Pupil size - - - - ↓ ↓ ↓ - - - Nystagmus ------Salivation ------Vocalization ------Pilo erection ------Micturition ------Irritability - - - + + + - - - - Disorientation /Staggering gate ------Head tap. aggressive ------Passivity - - - ↑+ ↑+ ↑+ - - - - Spontaneous activity - + ↓+ ↓+ ------Dec. motor activity - - - - - ↑+ ↑+ ↑+ - - Dec. resp. rate ------Dec. resp. depth ------Increase resp. rate ------Increase resp. depth ------Dyspnea ------Analgesia/pain response - - - - ↓+ ↓+ ↓+ - - - Anesthesia ------Corneal reflex ------Light reflex ------Ataxia ------Limb tone decrease ------Body tone - - - - ↓+ ↓+ ↓+ ↓+ - - Tail erection ------Tail lashing ------Enophthlmoses ------Exophthalmoses ------Touch response - - - - - ↓+ ↓+ - - - Tremor ------Straub tail ------Righting reflex - - - + + + + + - -

106

Table 39: Behavioural responses of V. thapsus fruit in mice

PARAMETERS V. thapsus fruit 500 mg/kg Control 0 min 5 min. 10 15 30 60min 2 hrs 4 hrs 24hrs min min min Lacrimation ------Pupil size - - - - ↓ ↓ ↓ - - - Nystagmus ------Salivation ------Vocalization ------Pilo erection ------Micturition ------Irritability ------Disorientation /Staggering gate ------Head tap. aggressive ------Passivity - - + + ↑+ - - - - - Spontaneous activity + + + + ------Dec. motor activity - - - - - + + ↑+ - - Dec. resp. rate ------Dec. resp. depth ------Increase resp. rate ------Increase resp. depth ------Dyspnea ------Analgesia/pain response - - - + ↓+ ↓+ ↓+ ↓+ - - Anesthesia ------Corneal reflex ------Light reflex - - - - - + + + - - Ataxia ------Limb tone decrease ------Body tone - - - - + + + + + - Tail erection - - - + + + - - - - Tail lashing ------Enophthlmoses ------Exophthalmoses ------Touch response - - + + + + + + - - Tremor ------Straub tail ------Righting reflex ------

107

Table 40: Behavioural responses of V. thapsus leave in mice

PARAMETERS V. thapsus leaves 100 mg/kg Control 0 min 5 min. 10 15 30 60min 2 hrs 4 hrs 24hrs min min min Lacrimation - - - - + ++ ++ - - - Pupil size - - ↑+ ↑+ ↓ ↓ ↓ - - - Nystagmus ------+ + - - Salivation ------Vocalization ------Pilo erection - - + + + + - - - - Micturition ------Irritability ------Disorientation /Staggering gate ------Head tap. aggressive ------Passivity - - ↑+ ↑+ ↑+ ↑ - - - - Spontaneous activity ↓+ ↓+ ↓+ ↓+ ------Dec. motor activity - - - - - ↑+ ↑+ ↑+ - - Dec. resp. rate ------Dec. resp. depth ------Increase resp. rate ------Increase resp. depth ------Dyspnea - - + + + + - - - - Analgesia/pain response - - - ↓+ ↓+ ↓+ ↓+ - - - Anesthesia ------Corneal reflex ------Light reflex ------Ataxia ------Limb tone decrease ------Body tone ------Tail erection ------Tail lashing ------Enophthlmoses ------Exophthalmoses - - - - - + + - - - Touch response - + + + + + + + + - Tremor - - + + + - - - - - Straub tail ------Righting reflex ------

108

Table 41: Behavioral responses of V. thapsus leave in mice

PARAMETERS V. thapsus leaves 300 mg/kg Control 0 min 5 min. 10 15 30 60min 2 hrs 4 hrs 24hrs min min min Lacrimation - - - - ++ ++ ++ - - - Pupil size - - ↑+ ↑+ ↓ ↓ ↓ - - - Nystagmus ------Salivation ------Vocalization ------Pilo erection - - + + + + - - - - Micturition ------Irritability ------Disorientation /Staggering gate ------Head tap. aggressive ------Passivity - - ↑+ ↑+ ↑+ ↑ - - - - Spontaneous activity ↓+ ↓+ ↓+ ↓+ ------Dec. motor activity - - - - - ↑+ ↑+ ↑+ - - Dec. resp. rate ------Dec. resp. depth ------Increase resp. rate ------Increase resp. depth ------Dyspnea - - + + ++ + + - - - Analgesia/pain response - - - ↓+ ↓++ ↓+ ↓+ - - - Anesthesia ------Corneal reflex ------Light reflex ------Ataxia ------Limb tone decrease ------Body tone ------Tail erection ------Tail lashing ------Enophthlmoses ------Exophthalmoses - - - - + + + - - - Touch response - + + + + + + + + - Tremor - - + + + - - - - - Straub tail ------Righting reflex - - - + ++ + + + + -

109

Table 42: Behavioral responses of V. thapsus leave in mice

PARAMETERS V. thapsus leaves 500 mg/kg Control 0 min 5 min. 10 15 30 60min 2 hrs 4 hrs 24hrs min min min Lacrimation - - - - + ++ ++ - - - Pupil size - - - - ↓ ↓ ↓ - - - Nystagmus ------+ + - - Salivation ------Vocalization ------Pilo erection - - + + + ++ + - - - Micturition ------Irritability ------Disorientation /Staggering gate ------Head tap. aggressive ------Passivity - - ↑+ ↑+ ↑+ ↑ - - - - Spontaneous activity ↓+ ↓+ ↓+ ↓+ ------Dec. motor activity - - - - + ↑+ ↑+ ↑+ - - Dec. resp. rate ------Dec. resp. depth ------Increase resp. rate ------Increase resp. depth ------Dyspnea - - + + + + - - - - Analgesia/pain response - - - ↓+ ↓+ ↓+ ↓+ - - - Anesthesia ------Corneal reflex ------Light reflex ------Ataxia ------Limb tone decrease ------Body tone ------Tail erection ------Tail lashing ------Enophthlmoses ------Exophthalmoses - - - - - + + - - - Touch response - + + + + + + + + - Tremor - - + + + - - - - - Straub tail ------Righting reflex - + + + + + + + + -

110

Table 43: Behavioral responses of V. thapsus root in mice

PARAMETERS V. thapsus root 100 mg/kg Control 0 min 5 min. 10 15 30 60min 2 hrs 4 hrs 24hrs min min min Lacrimation ------Pupil size - - - - - ↓ ↓ - - - Nystagmus ------Salivation ------Vocalization ------Pilo erection ------Micturition ------Irritability - - - + + + - - - - Disorientation /Staggering gate ------Head tap. aggressive ------Passivity - - - - ↓+ - - - - - Spontaneous activity + + + - - - - Dec. motor activity - - - - - ↓+ ↓+ ↓+ - - Dec. resp. rate ------Dec. resp. depth ------Increase resp. rate ------Increase resp. depth ------Dyspnea ------Analgesia/pain response - - - - - ↓+ ↓+ - - - Anesthesia ------Corneal reflex ------Light reflex - - - - - + + + - - Ataxia ------Limb tone decrease ------Body tone - - - - + + - - - - Tail erection - - - + + + - - - - Tail lashing ------Enophthlmoses ------Exophthalmoses ------Touch response - - + + + + - - Tremor ------Straub tail ------Righting reflex - - - - - + + + - -

111

Table 44: Behavioral responses of V. thapsus root in mice

PARAMETERS V. thapsus root 300 mg/kg Control 0 min 5 min. 10 15 30 60min 2 hrs 4 hrs 24hrs min min min Lacrimation ------Pupil size - - - - ↓ ↓ - - - Nystagmus ------Salivation ------Vocalization ------Pilo erection ------Micturition ------Irritability - - + + + ++ - - - - Disorientation /Staggering gate ------Head tap. aggressive ------Passivity - - - + ↓+ - - - - - Spontaneous activity + + + ++ - - - - Dec. motor activity - - - - ↓+ ↓+ ↓+ ↓+ - - Dec. resp. rate ------Dec. resp. depth ------Increase resp. rate ------Increase resp. depth ------Dyspnea ------

Analgesia/pain response - - - ↓+ ↓+ ↓++ ↓+ - - - Anesthesia ------Corneal reflex ------Light reflex - - - - + + + + - - Ataxia ------Limb tone decrease ------Body tone - - - - + + - - - - Tail erection - - + + + + + - - - Tail lashing ------Enophthlmoses ------Exophthalmoses ------Touch response - - + + + + + - - Tremor ------Straub tail ------Righting reflex - - - - + + + + - -

112

Table 45: Behavioral responses of V. thapsus root in mice

PARAMETERS V. thapsus root 500 mg/kg Control 0 min 5 min. 10 15 30 60min 2 hrs 4 hrs 24hrs min min min Lacrimation ------

Pupil size - - - - +↓ ↓ ↓ - - - Nystagmus ------Salivation ------Vocalization ------Pilo erection ------Micturition ------Irritability - - + + + + + - - - Disorientation /Staggering gate ------Head tap. aggressive ------Passivity - - - + ↓+ + - - - - Spontaneous activity + ++ + + - - - Dec. motor activity - - - ↓+ ↓+ ↓+ ↓+ ↓+ - - Dec. resp. rate ------Dec. resp. depth ------Increase resp. rate ------Increase resp. depth ------Dyspnea ------Analgesia/pain response - - - ↓+ ↓+ ↓+ ↓+ ↓+ - - Anesthesia ------Corneal reflex ------Light reflex - - - - - + + + - - Ataxia ------Limb tone decrease ------Body tone - - - - + + + - - - Tail erection ------Tail lashing ------Enophthlmoses ------Exophthalmoses ------Touch response - - + + + + + + - Tremor ------Straub tail ------Righting reflex - - - + + + + + - -

113

Table 46: Behavioural responses of V. thapsus stem in mice

PARAMETERS V. thapsus stem 100 mg/kg Control 0 min 5 min. 10 15 30 60min 2 hrs 4 hrs 24hrs min min min Lacrimation ------Pupil size ------Nystagmus ------Salivation ------Vocalization ------Pilo erection ------Micturition ------Irritability - - - + + + - - - - Disorientation /Staggering gate ------Head tap. aggressive ------Passivity - - ↓+ ↓+ ↓+ - - - - - Spontaneous activity + + + + ------Dec. motor activity - - - - - ↓+ ↓+ ↓+ - - Dec. resp. rate ------Dec. resp. depth ------Increase resp. rate - - - - - + + - - - Increase resp. depth ------Dyspnea ------Analgesia/pain response - - - ↓+ ↓+ ↓+ ↓+ - - - Anesthesia - - - - + + - - - - Corneal reflex ------Light reflex - - - - - + + + - - Ataxia ------Limb tone decrease ------Body tone - - - - + + + - - - Tail erection - - - - + + - - - - Tail lashing ------Enophthlmoses ------Exophthalmoses ------Touch response - - - + + + + + - - Tremor ------Straub tail ------Righting reflex ------

114

Table 47: Behavioral responses of V. thapsus stem in mice

PARAMETERS V. thapsus stem 300 mg/kg Control 0 min 5 min. 10 15 30 60min 2 hrs 4 hrs 24hrs min min min Lacrimation ------Pupil size ------Nystagmus ------Salivation ------Vocalization ------Pilo erection ------Micturition ------Irritability - - - + + + + - - - Disorientation /Staggering gate ------Head tap. aggressive ------Passivity - - - - ↓+ - - - - - Spontaneous activity ++ ++ + + ------Dec. motor activity - - - - - ↓+ ↓+ ↓+ - - Dec. resp. rate ------Dec. resp. depth ------Increase resp. rate - - - + + + - - - - Increase resp. depth - - - + + + - - - - Dyspnea ------Analgesia/pain response - - - ↓+ ↓+ ↓+ ↓+ - - - Anesthesia ------Corneal reflex - - - + + + - - - - Light reflex - - - + + + + - - - Ataxia ------Limb tone decrease ------Body tone - - - + + + + + - - Tail erection - - - + + + - - - - Tail lashing ------Enophthlmoses ------Exophthalmoses ------Touch response - - - - + + + - - - Tremor ------Straub tail ------Righting reflex ------

115

Table 48: Behavioral responses of V. thapsus stem in mice

PARAMETERS V. thapsus stem 500 mg/kg Control 0 min 5 min. 10 15 30 60min 2 hrs 4 hrs 24hrs min min min Lacrimation ------Pupil size ------Nystagmus ------Salivation ------Vocalization ------Pilo erection ------Micturition ------Irritability - - + + + ++ + - - - Disorientation /Staggering gate ------Head tap. aggressive ------Passivity - - - - ↓+ + - - - - Spontaneous activity ++ ++ ++ ++ ------Dec. motor activity - - - - - ↓+ ↓+ ↓+ - - Dec. resp. rate ------Dec. resp. depth ------Increase resp. rate - - + ++ ++ ++ - - - - Increase resp. depth - - + + + + - - - - Dyspnea ------Analgesia/pain response - - - ↓+ ↓+ ↓+ ↓+ - - - Anesthesia ------Corneal reflex - - - + + + - - - - Light reflex - - - + + + + - - - Ataxia ------Limb tone decrease ------Body tone - - - + + + + + - - Tail erection ------Tail lashing ------Enophthlmoses ------Exophthalmoses ------Touch response - - - + + + + - - - Tremor ------Straub tail ------Righting reflex ------

116

Table 49: Assessment of neuropharmacological activity in 30 minutes

(Open field and Head dip activity )

Treatment Dose mg/kg Open field activity Head dip activity orally (Mean no. of (Mean no. of observations ± S.E.M) observations ±S.E.M) Control 0.5 ml 287 + 2.62 43.8 + 3.90 saline R. f fruit 100 mg/kg 474.2±2.44** 34.8±2.48 300 mg/kg 404 ±1.66** 26.6 ± 2.92* 500 mg/kg 435 ± 1.94* 16.4 ± 2.28** R. f leaves 100 mg/kg 255.4±2.74 11.8±2.32** 300 mg/kg 259.4 ± 1.29 17.6 ± 1.97** 500 mg/kg 306.2± 2.54 15.4 ± 2.19** R. f root 100 mg/kg 422.2±0.58** 14.6±1.73** 300 mg/kg 358.4± 2.21** 9.6±1.94** 500 mg/kg 306.6 ± 1.92** 11 ± 1.79** R. f stem 100 mg/kg 293.8±1.47 33.2 ± 0.86 300 mg/kg 197.6 ± 1.51** 19 ± 1.27** 500 mg/kg 186.8 ± 2.09** 32.4 + 1.21 Diazepam 2 mg/kg 35 ± 0.89 ** 22 ± 1.03 ** Imipramine 15 mg/kg 302 + 1.21 83 ± 1.33** Caffeine 10 mg/kg 271 ± 1.38 75 ± 3.43** Mean ±S.E.M; n = 5; Significance with respect to control. (* = Significant results, ** = Highly significant results)

117

Table 50: Assessment of neuropharmacological activity in 30 minutes

(Open field and Head dip activity)

Treatment Dose mg/kg Open field activity Head dip activity orally (Mean no. of (Mean no. of observations ± S.E.M) observations ±S.E.M) Control 0.5 ml saline 287 + 2.62 43.8 + 3.90 V. t fruit 100 mg/kg 298.2 ± 3.82 22.4 ± 3.71* 300 mg/kg 322.6 ± 2.76** 16.2 ± 2.66** 500 mg/kg 361.8±2.32** 8.2±2.48** V. t leaves 100 mg/kg 108±2.52** 18±2.63** 300 mg/kg 114 ± 2.60** 10.4 ± 2.86** 500 mg/kg 117± 1.87** 9.2 ±1.99** V. t root 100 mg/kg 156±2.85** 26.6±2.78* 300 mg/kg 471.8± 3.19** 21.2±2.18* 500 mg/kg 475 ± 1.52** 44.2 ± 1.50 V. t stem 100 mg/kg 498.4±2.55** 27.8 ± 2.93 300 mg/kg 490.6 ± 2.81** 27.4 ± 0.93 500 mg/kg 469.4 ±2.63** 23.2 + 2.75* Diazepam 2 mg/kg 35 ± 0.89 ** 22 ± 1.03 ** Imipramine 15 mg/kg 302 + 1.21 83 ± 1.33** Caffeine 10 mg/kg 271 ± 1.38 75 ± 3.43** Mean ±S.E.M; n = 5; Significance with respect to control. (* significant ** highly significant)

118

Table 51: Assessment of neuropharmacological activity (Exploratory activity)

Treatment Dose mg/kg Cage cross activity Rearing activity orally (Mean no. of (Mean no. of observations ± S.E.M) observations ± S.E.M) Control 0.5 ml 65.4 ±4.13 50.6 ± 1.53 Saline R. f fruit 100 mg/kg 65.8±2.81 34±1.21 300 mg/kg 70±1.71 46±1.43 500 mg/kg 90.6±1.64** 55.4±1.61 R. f leaves 100 mg/kg 44.6±1.87* 28±1.51 300 mg/kg 62.6 ± 2.12 41±1.11 500 mg/kg 71.4 ±1.40 57.5 ± 0.81 R. f root 100 mg/kg 56.4±1.17 23.5±1.15 300 mg/kg 52.2 ± 1.86* 33.8± 0.81 500 mg/kg 46.2 ± 1.78** 18.6 ± 1.43 R. f stem 100 mg/kg 65.8±2.34 24±1.42 300 mg/kg 50.2±2.18* 36.4±1.13 500 mg/kg 82.2±1.94* 45.4±0.83 Diazepam 2 mg/kg 41 ± 2.03 07 ± 0.82** Imipramine 15 mg/kg 75 ± 2.0** 55.2 ± 1.33 Caffeine 15 mg/kg 34 ± 1.89* 11.6± 0.93* Mean + S.E.M; n = 5; Significance with respect to control. (* = Significant results, ** = Highly significant results)

119

Table 52: Assessment of neuropharmacological activity (Exploratory activity)

Treatment Dose mg/kg Cage cross activity Rearing activity orally (Mean no. of (Mean no. of observations ± S.E.M) observations ± S.E.M) Control 0.5 ml 65.40 ±4.13 50.6 ± 1.53 Saline V. t fruit 100 mg/kg 42.8±0.97 14±0.81 300 mg/kg 57±0.90 19.4±1.43 500 mg/kg 40.2±0.67 16±1.10 V. t leaves 100 mg/kg 11.2±0.38** 07±0.51 300 mg/kg 39.2 ± 1.78** 19±1.21 500 mg/kg 59.2 ±1.69 26.4 ± 1.21 V. t root 100 mg/kg 26.6±0.93* 18±1.31 300 mg/kg 21.1 ± 0.86** 13.8± 0.76 500 mg/kg 44.2 ± 1.50** 19.6 ± 1.03 V. t stem 100 mg/kg 78±0.95 24±1.12 300 mg/kg 74.8±1.60 19.4±1.33 500 mg/kg 55.2±0.49 14.4±1.03 Diazepam 2 mg/kg 41 ± 2.03 07 ± 0.82** Imipramine 15 mg/kg 75 ± 2.0** 55.2 ± 1.33 Caffeine 15 mg/kg 34 ± 1.89* 11.6± 0.93* Mean + S.E.M; n = 5; Significance with respect to control. (* = Significant results, ** = Highly significant results)

120

Table 53: Assessment of neuropharmacological activity (Traction time)

Traction time with ± SEM (Time in sec) Group 0 30 60 90 120 150 210 270 min min min min min min min Control 9.4± 9.8± 10.05 9.8± 10.0± 10.02 ± 9.5± 10.09 0.76 1.11 ± 1.12 1.09 2.08 1.02 0.89 ± 1.21 R. f fruit 09± 9.2 ± 9.6± 9.7 ± 10± 9.8 ± 9.3± 8.8 ± 100 mg/kg 0.86 1.21 0.61 0.63 0.55 0.51 0.33 0.50 R. f fruit 8.7± 8.5 ± 9.6± 9.4 ± 9.7± 9.7 ± 9.3± 9.4 ± 300 mg/kg 0.41 0.35 1.20 0.83 0.51 0.41 0.21 0.50 R. f fruit 08± 8.2 ± 7.3*± 7.3* ± 7.0*± 7.4* ± 8± 8.4 ± 500 mg/kg 1.11 0.18 1.03 0.53 0.57 0.21 0.27 0.46 R. f leaves 9.2± 9.4 ± 9.8± 9.9 ± 10.2± 10 ± 9.5± 9 ± 100 mg/kg 0.46 1.31 0.71 0.73 0.65 0.61 0.43 0.60 R. f leaves 8.9± 8.7 ± 9.8± 9.6 ± 9.9± 9.9 ± 9.5± 9.6 ± 300 mg/kg 0.51 0.45 1.30 0.93 0.61 0.51 0.31 0.60 R. f leaves 8.5± 8.7 ± 7.8*± 7.8* ± 7.5*± 7.9*± 8.5± 8.9 ± 500 mg/kg 1.21 0.28 1.13 0.63 0.67 0.31 0.37 0.56 R. f root 9.1± 9.3 ± 9.6± 9.8 ± 9.8± 10 ± 9.5± 9.8 ± 100 mg/kg 0.82 1.20 0.63 0.61 0.54 0.52 0.30 0.50 R. f root 8.8± 9.00 ± 9.8± 9.6 ± 9.5± 9.8 ± 9.4± 9.7 ± 300 mg/kg 0.45 0.65 1.70 0.63 0.31 0.48 0.71 0.59 R. f root 08.4± 8.8 ± 8.3± 9.0 ± 9.0± 9.4 ± 9.3± 9.4 ± 500 mg/kg 1.26 0.39 1.23 0.58 0.77 0.41 0.29 0.66 R. f stem 9.3± 9.7± 10.0 ± 9.6± 9.8± 10.0 ± 9.2± 10.0 ± 100 mg/kg 0.56 1.22 1.32 1.29 1.20 1.42 0.79 1.23 R. f stem 9.0± 9.5± 9.9 ± 9.4± 9.6± 10.0 ± 9.5± 9.2 ± 300 mg/kg 0.53 1.33 1.20 1.25 1.33 1.66 0.73 1.20 R. f stem 9.0± 9.4± 9.7 ± 9.5± 9.3± 9.7 ± 9.2± 9.0 ± 500 mg/kg 0.44 1.27 0.72 0.29 1.38 0.40 0.83 1.08 Diazepam 11 ± 12.2* 14.4* 17** 17.7** 18.2**± 15** 14.8* 2 mg/kg 0.21 ± 0.35 ± 0.82 ± 0.32 ±1.31 0.66 ± 0.72 ± 0.71 Imipramine 8.2± 8.4 ± 9± 8.4 ± 9.2± 9 ± 8.5± 8.2 ± 15 mg/kg 0.21 1.23 2.07 1.22 0.72 0.53 0.64 0.80 Mean ± S.E.M; n = 5; Significance with respect to control. * = Significant results, ** = Highly significant results)

121

Table 54: Assessment of neuropharmacological activity (Traction time)

Traction time with ± SEM (Time in sec) Group 0 30 60 90 120 150 210 270 min min min min min min min Control 9.4± 9.8± 10.05 9.8± 10.0± 10.02 ± 9.5± 10.09 0.76 1.11 ± 1.12 1.09 2.08 1.02 0.89 ± 1.21 V. t fruit 09± 8.8 ± 8.6± 9.4 ± 9.3± 9.0 ± 9.5± 9 ± 100 mg/kg 0.65 1.42 0.46 0.67 0.65 0.51 0.45 0.84 V. t fruit 08± 7.4* ± 7.4*± 8.4 ± 9.4± 9.4 ± 9.4± 10 ± 300 mg/kg 0.45 0.56 1.40 0.64 0.31 0.60 0.58 0.39 V. t fruit 08± 06** 6.6** 7.5* 9.4± 9.5 ± 9.5± 9.7 ± 500 mg/kg 0.49 ± 0.66 ± 1.8 ± 0.94 0.82 0.26 0.38 0.37 V. t leaves 8.8± 8.6 ± 8.4± 9.3 ± 9.1± 8.8 ± 9.2± 9 ± 100 mg/kg 0.35 1.30 0.56 0.37 0.75 0.59 0.69 0.34 V. t leaves 8.2± 7.2 *± 7.2*± 8.0 ± 9.2± 9.2 ± 9.2± 9 ± 300 mg/kg 0.45 0.76 1.55 0.82 0.37 0.66 0.50 0.80 V. t leaves 08± 7.0** 6.2**± 7.2* ± 8.4± 8.5 ± 9.0± 9.2 ± 500 mg/kg 0.91 ± 0.44 1.32 0.90 0.72 0.56 0.58 0.77 V. t root 9.5± 9.4± 9.2 ± 9.5± 9.2± 9.4 ± 9.4± 9.9 ± 100 mg/kg 1.40 1.12 1.5 1.43 1.48 1.77 0.72 1.32 V. t root 9.2 ± 9.2 ± 9.0 ± 9.0 ± 9.6 ± 9.4± 9.0± 10.0 300 mg/kg 1.14 0.57 0.64 0.41 1.68 0.50 1.19 ±1.12 V. t root 9.5± 8.5 ± 8.2± 8.2 ± 8.0± 9.0 ± 8.5± 9.2 ± 500 mg/kg 0.79 1.27 1.28 1.70 0.11 0.51 0.49 0.38 V. t stem 10± 9.8± 9.25 ± 10± 10.2± 9.8 ± 9.8± 9.09 ± 100 mg/kg 0.74 1.31 1.35 1.49 1.03 1.42 0.82 1.71 V. t stem 9.5 ± 9.4 ± 9.2 ± 9.0 ± 9.5 ± 9.2± 9.2± 9.4 300 mg/kg 0.52 0.87 0.44 0.55 1.60 0.57 1.17 ±1.12 V. t stem 9.5± 8.7 ± 8.5± 8.5 ± 9.0± 9.2 ± 9± 9.2 ± 500 mg/kg 0.82 1.22 1.18 1.70 0.81 0.55 0.41 0.78 Diazepam 11 ± 12.2* 14.4* 17** 17.7** 18.2**± 15** 14.8* 2 mg/kg 0.21 ± 0.35 ± 0.82 ± 0.32 ±1.31 0.66 ± 0.72 ± 0.71 Imipramine 8.2± 8.4 ± 9± 8.4 ± 9.2± 9 ± 0.53 8.5± 8.2 ± 15 mg/kg 0.21 1.23 2.07 1.22 0.72 0.64 0.80 Mean ± S.E.M; n = 5; Significance with respect to control. * = Significant results, ** = Highly significant results)

122

Table 55: Assessment of neuropharmacological activity (Forced swimming test) Treatment Dose mg/kg Mobility time Immobility time orally (Mean no. of (Mean no. of observations ± S.E.M) Observations ± S.E.M) Control 0.5ml Saline 3:45 ± 0.059 2:15 ± 0.059 R. f fruit 100 mg/kg 1:14±0.0323** 4:46±0.0323** 300 mg/kg 0:25 ± 0.062** 5:35 ± 0.062** 500 mg/kg 1:25 ± 0.0338* 4:35 ± 0.0338* R. f leaves 100 mg/kg 2:27±0.059 3:33±0.059 300 mg/kg 2:22 ± 0.076 3:38 ± 0.076 500 mg/kg 2:04 ± 0.028* 3:56 ± 0.028* R. f root 100 mg/kg 1:19±0.0193** 4:41±0.0193** 300 mg/kg 2:07±0.0341* 3:53±0.0341* 500 mg/kg 1:11±0.2016** 4:49±0.2016** R. f stem 100 mg/kg 2:28±0.0316 3:32±0.0316 300 mg/kg 2:03 ± 0.030* 3:57 ± 0.030* 500 mg/kg 3:03 ± 0.0353 2:57 ± 0.0353 Diazepam 2 mg/kg 1:55 ± 0.08** 4:05 ± 0.08** Imipramine 15 mg/kg 3:45±0.09 2:15 ± 0.09 Mean + S.E.M; n = 5; Significance with respect to control. (* = Significant results, ** = Highly significant results)

123

Table 56: Assessment of neuropharmacological activity (Forced swimming test)

Treatment Dose mg/kg Mobility time Immobility time orally (Mean no. of (Mean no. of Observations ± S.E.M) Observations ± S.E.M) Control 0.5ml Saline 3:45 ± 0.059 3:45 ± 0.059 V. t fruit 100 mg/kg 1:36±0.0285** 4:24±0.0285** 300 mg/kg 2:09 ± 0.0336* 3:51 ± 0.0336* 500 mg/kg 1:40 ± 0.0296** 4:20 ± 0.0296** V. t leaves 100 mg/kg 1:30±0.0236** 4:30±0.0236** 300 mg/kg 0:36 ± 0.0283** 5:36 ± 0.0283** 500 mg/kg 2:20 ± 0.0296* 3:40 ± 0.0296* V. t root 100 mg/kg 2:40±0.0296* 3:20±0.0296* 300 mg/kg 3:07±0.0418 2:53±0.0148 500 mg/kg 2:58±0.0273 3:02±0.0273 V. t stem 100 mg/kg 2:51±0.0303 3:09±0.0303 300 mg/kg 2:39 ± 0.026* 3:21 ± 0.026* 500 mg/kg 3:34 ± 0.0238 2:26 ± 0.0238 Diazepam 2 mg/kg 1:55 ± 0.08 ** 4:05 ± 0.08 ** Imipramine 15 mg/kg 3:45±0.09 2:15 ± 0.09 Mean + S.E.M; n = 5; Significance with respect to control. (* = Significant results, ** = Highly significant results)

124

Table 57: Antibacterial activities of R. fruticosus fruit (zone of inhibitions in mm with ± SEM, disc size: 8mm)

Dose µg E. coli S. typhi S. P. M. Citrobacter B. P. /disc aureus mirabilis luteus subtilis aeruginosa 20 0±0 0±0 8.1±0.16 0±0 8±0.08 8±0.14 8±0.09 8±0.11 40 0±0 0±0 8±0.17 0±0 8±0.12 8±0.11 8±0.10 8±0.12 60 0±0 0±0 8±0.13 0±0 8±0.09 8±0.17 8±0.08 8±0.09 80 0±0 0±0 8±0.12 0±0 8±0.16 8±0.12 8±0.10 9±0.2 100 0±0 0±0 8±0.2 0±0 8±0.09 10±0.11 8±0.09 10±0.18 1000 8±0.08 10±0.2 10±0.22 0±0 9±0.19 11±0.21 9±0.2 10±0.11 2000 9±0.09 11±0.2 11±0.23 8±0.09 11±0.18 12±0.10 11±0.09 11±0.26 MIC 1000 1000 20 2000 20 20 20 20 Gentamicin 19±0.8 20±0.14 19±0.65 19±0.2 19±0.2 17±0.55 16±0.09 19±0.11 20µg 4 Ampicillin 23±0.1 15±0.11 21±0.55 15±0.09 21±0.67 16±0.62 22±0.17 16±0.22 1mg 1 Amoxicillin 24±0.7 20±0.03 20±0.06 22±0.07 20±0.91 18±0.13 20±0.18 22±0.23 1mg 8

Table 58: Antibacterial activity of methanolic extract of R. fruticosus leaves (zone of inhibitions in mm with ± SEM, disc size: 8mm)

Dose µg E. coli S. typhi S. P. M. Citrobacter B. P. /disc aureus mirabilis luteus subtilis aeruginosa 20 8±0.13 0±0 8.2±0.10 0±0 0±0 0±0 0±0 0±0 40 10±0.09 0±0 9±0.12 9±0.09 0±0 0±0 0±0 0±0 60 11±0.12 0±0 8±0.10 9±0.19 0±0 0±0 0±0 0±0 80 12±0.02 0±0 8±0.2 10±0.13 0±0 0±0 0±0 0±0 100 11±0.11 0±0 9±0.21 9±0.13 8±0.07 0±0 8±0.05 0±0 1000 11±0.19 8±0.09 8±0.08 10±0.09 9±0.15 11±0.12 9±0.1 10±0.16 2000 11±0.12 9±0.09 10±0.16 10±0.09 10±0.10 12±0.11 10±0.05 10±0.18 MIC 20 1000 20 40 100 1000 40 1000 Gentamicin 19±0.84 20±0.14 19±0.65 19±0.2 19±0.2 17±0.55 16±0.09 19±0.11 20µg Ampicillin 23±0.11 15±0.11 21±0.55 15±0.09 21±0.67 16±0.62 22±0.17 16±0.22 1mg Amoxicillin 24±0.78 20±0.03 20±0.06 22±0.07 20±0.91 18±0.13 20±0.18 22±0.23 1mg

125

Table 59: Antibacterial activity of R. fruticosus root (zone of inhibitions in mm with ± SEM, disc size: 8mm)

Dose µg E. coli S. typhi S. P. M. Citrobacter B. P. /disc aureus mirabilis luteus subtilis aeruginosa 20 8.7±0.11 9.2±0.0 8±0.17 8±0.12 8±0.16 0±0 10±0.11 0±0 9 40 9±0.12 9±0.11 8±0.12 8±0.17 8±0.13 0±0 10±0.16 9±0.11 60 9±0.09 9±0.12 8±0.10 9±0.12 9±0.16 8±0.09 9±0.19 9±0.2 80 10±0.10 9±0.15 8±0.08 13±0.10 13±0.1 11±0.30 10±0.13 11±0.11 100 10±0.30 8±0.09 8±0.11 13±0.2 13±0.20 9±0.09 9±0.15 10±0.09 1000 13±0.13 12±0.13 11±0.11 17±0.10 17±0.10 12±0.19 10±0.21 12±0.12 2000 13±0.08 13±0.09 12±0.13 17±0.12 17±0.17 13±0.23 12±0.20 12.5±0.13 MIC 20 20 20 20 60 20 20 40 Gentamicin 19±0.84 20±0.14 19±0.65 19±0.2 19±0.2 17±0.55 16±0.09 19±0.11 20µg Ampicillin 23±0.11 15±0.11 21±0.55 15±0.09 21±0.67 16±0.62 22±0.17 16±0.22 1mg Amoxicillin 24±0.78 20±0.03 20±0.06 22±0.07 20±0.91 18±0.13 20±0.18 22±0.23 1mg

Table 60: Antibacterial activity of R. fruticosus stem (zone of inhibitions in mm with ± SEM, disc size: 8mm)

Dose µg E. coli S. typhi S. P. M. Citrobacter B. P. /disc aureus mirabilis luteus subtilis aeruginosa 20 9.2±0.15 8.9±0.1 8.8±0.11 9.8±0.09 8±0.12 9±0.11 8±0.17 9±0.15 40 9±0.11 8±0.08 8.5±0.09 10±0.12 9±0.10 8±0.10 9±0.11 9.5±0.13 60 8±0.10 9±0.09 9.5±0.14 8±0.12 8±0.11 8±0.10 8±0.11 9±0.19 80 9±0.09 9±0.12 9±0.17 8±0.2 13±0.1 8±0.09 8±0.11 9±0.20 100 8±0.2 10±0.09 8.5±0.19 10±0.16 8±0.10 8±0.12 9±0.2 11±0.12 1000 11±0.11 10±0.11 10±0.21 15±0.17 11±0.2 11±0.15 10±0.19 11±0.24 2000 11±0.2 11±0.13 11±0.12 15±0.19 11±0.12 12±0.2 11±0.17 11±0.10 MIC 20 20 20 20 20 20 20 20 Ampicillin 23±0.11 15±0.11 21±0.55 15±0.09 21±0.67 16±0.62 22±0.17 16±0.22 1mg Amoxicillin 24±0.78 20±0.03 20±0.06 22±0.07 20±0.91 18±0.13 20±0.18 22±0.23 1mg

126

Table 61: Antibacterial activity of V. thapsus fruit (zone of inhibitions in mm with ± SEM, disc size: 8mm)

Dose µg E. coli S. typhi S. aureus P. M. Citrobacter B. P. /disc mirabilis luteus subtilis aeruginosa 20 0±0 0±0 0±0 0±0 0±0 0±0 0±0 0±0 40 0±0 0±0 0±0 0±0 0±0 0±0 0±0 0±0 60 0±0 0±0 0±0 0±0 0±0 0±0 0±0 0±0 80 0±0 0±0 0±0 0±0 0±0 0±0 0±0 0±0 100 0±0 0±0 0±0 0±0 0±0 0±0 0±0 0±0 1000 9±0.30 9±0.2 9±0.11 11±0.12 11±0.15 8±0.11 9±0.11 14±0.19 2000 10±0.22 10±0.36 9.7±0.14 15±0.19 12±0.20 8±0.18 10±0.05 16±0.11 MIC 1000 1000 1000 1000 1000 1000 1000 1000 Ampicillin 23±0.11 15±0.11 21±0.55 15±0.09 21±0.67 16±0.62 22±0.17 16±0.22 1mg Amoxicillin 24±0.78 20±0.03 20±0.06 22±0.07 20±0.91 18±0.13 20±0.18 22±0.23 1mg

Table 62: Antibacterial activity of V. thapsus leaves (zone of inhibitions in mm with ± SEM, disc size: 8mm)

Dose µg E. coli S. typhi S. P. M. Citrobacter B. P. /disc aureus mirabilis luteus subtilis aeruginosa 20 0±0 0±0 8±0.12 8±0.12 0±0 0±0 8±0 0±0 40 0±0 0±0 8±0.17 8±0.20 0±0 0±0 8±0 8±0.09 60 0±0 0±0 9±0.19 8±0.09 8±0.09 8±0.11 8±0 8±0.11 80 0±0 0±0 9±0.20 9±0.15 8±0.08 8±0.12 8±0 8±0.22 100 0±0 0±0 9±0.11 9±0.23 8±0.15 8±0.16 8±0 9±0.23 1000 8±0.10 8±0.09 9±0.09 9±0.22 9±0.13 9±0.17 10±0.11 10±0.16 2000 9±0.08 9±0.14 9±0.09 10±0.07 9±0.25 9±0.20 10±0.05 11±0.18 MIC 1000 1000 20 20 60 60 20 40 Ampicillin 23±0.11 15±0.11 21±0.55 15±0.09 21±0.67 16±0.62 22±0.17 16±0.22 1mg Amoxicillin 24±0.78 20±0.03 20±0.06 22±0.07 20±0.91 18±0.13 20±0.18 22±0.23 1mg

127

Table 63: Antibacterial activity of V. thapsus root (zone of inhibitions in mm with ± SEM, disc size: 8mm)

Dose µg E. coli S. typhi S. P. M. Citrobacter B. P. /disc aureus mirabilis luteus subtilis aeruginosa 20 0±0 0±0 0±0 0±0 0±0 0±0 0±0 0±0 40 0±0 0±0 0±0 0±0 0±0 0±0 0±0 0±0 60 0±0 0±0 0±0 0±0 0±0 0±0 0±0 0±0 80 0±0 0±0 0±0 0±0 0±0 0±0 9.2±0.11 0±0 100 0±0 0±0 0±0 0±0 0±0 0±0 9±0.07 0±0 1000 0±0 10±0.25 9±0.09 0±0 0±0 8.4±0.09 9.3±0.14 8±0.19 2000 0±0 11±0.15 9±0.11 9.5±0.1 9.7±0.2 9.3±0.23 11±0.05 8.2±0.28 MIC 0 1000 1000 2000 2000 1000 80 1000 Ampicillin 23±0.11 15±0.11 21±0.55 15±0.09 21±0.67 16±0.62 22±0.17 16±0.22 1mg Amoxicillin 24±0.78 20±0.03 20±0.06 22±0.07 20±0.91 18±0.13 20±0.18 22±0.23 1mg

Table 64: Antibacterial activity of V. thapsus stem (zone of inhibitions in mm with ± SEM, disc size: 8mm)

Dose µg E. coli S. typhi S. P. M. Citrobacter B. P. /disc aureus mirabilis luteus subtilis aeruginosa 20 8±0.14 0±0 0±0 0±0 0±0 8±0.11 0±0 0±0 40 8±0.23 0±0 0±0 0±0 0±0 8±0.17 0±0 0±0 60 8±0.17 0±0 0±0 0±0 0±0 8±0.13 0±0 0±0 80 8±0.31 0±0 0±0 0±0 0±0 8±0.17 0±0 0±0 100 8±0.12 0±0 0±0 0±0 0±0 8±0.24 0±0 0±0 1000 9±0.22 0±0 0±0 0±0 9±0.12 9±0.28 0±0 0±0 2000 9±0.24 8±0.05 0±0 0±0 9±0.17 9.5±0.08 0±0 0±0 MIC 20 2000 0 0 1000 20 0 0 Ampicillin 23±0.11 15±0.11 21±0.55 15±0.09 21±0.67 16±0.62 22±0.17 16±0.22 1mg Amoxicillin 24±0.78 20±0.03 20±0.06 22±0.07 20±0.91 18±0.13 20±0.18 22±0.23 1mg

128

Table 65: Antifungal activity of R. fruticosus and V. thapsus different parts extracts, disc size: 8mm)

Treatment S. A. T. M. C. F. A. A. cerevisiae parasiticus rubrum phaseolinia albican solani niger effusus

R. f fruit 0 0 0 0 0 0 0 0 R. f leaves 0 0 0 0 0 0 0 0 R. f root 0 0 0 0 0 0 0 0 R. f stem 0 0 0 0 0 0 0 0 V. t fruit 0 0 0 0 0 0 0 0 V. t leaves 0 0 0 0 0 0 0 0 V. t root 0 0 0 0 0 0 0 0 V. t leaves 0 0 0 0 0 0 0 0 Itraconazole 19±0.67 16±0.88 21±0.63 16.5±0.31 14±0.66 12±0.34 12±0.14 14±0.25 2mg Amphoteracin 14±0.91 13±0.71 11±0.97 15±0.54 14±0.54 12±0.44 18±0.31 13±0.24 B 2mg Doses 10, 20, 25, 50, 100 and 200µg/disc were applied for each sample

Table 66: Insecticide activity of R. fruticosus fruit

Dose Tribolium castaneum Sitophilus oryzae No. of Time of onset of drug % Time of onset of % survivor action (immobility Mortality drug action Mortality time) (immobility time ) 1 mg 10 10min; 1P, 30min; 1D 10 - 0 5 mg 10 30min; 2P, 12hr; 1D 10 1hr; 1P 0 10 mg 10 1hr; 1P 0 30min; 1P 0 25 mg 10 24hr; 1P &1D 10 2hr; 1P, 2hr; 1D 10 50 mg 10 30min; 2P, 1hr; 1D 10 3hr; 1P, 4hr; 1D 10 75 mg 10 10min; 1P 0 - 0 100 mg 10 5min; 1P 0 10min; 1P 0 Name of Insects % Mortality + ve Control Permethrin - ve Control (Coopex) at conc. 235.9 (solvent) µg/cm 2 Tribolium castaneum 100 0 Sitophilus oryzae 100 0

R = repellent, P = paralyzed, D= died

129

Table 67: Insecticide activity of R. fruticosus leaves

Dose Tribolium castaneum Sitophilus oryzae No. of Time of onset of % Time of onset % survivor drug action Mortality of drug action Mortality (immobility time) (immobility time) 1 mg 10 - 0 - 0 5 mg 10 - 0 - 0 10 mg 10 1hr; 01 P, 24hr; 1D 10 - 0 25 mg 10 2hr; 01 P&24hr; 1D 10 10hr; 1P 0 50 mg 10 24hr; 1P and 1D 10 1hr; 2P 0 75 mg 10 30min; 1P & 1D 10 1hr; 1P 0 100 mg 10 1hr; 1P, 2R 0 12hr; 1D, 1R 10 Name of Insects % Mortality + ve Control Permethrin - ve Control (Coopex) at conc. 235.9 (solvent) µg/cm 2 Tribolium castaneum 100 0 Sitophilus oryzae 100 0

R = repellent, P = paralyzed, D= died

Table 68: Insecticide activity of R. fruticosus root

Dose Tribolium castaneum Sitophilus oryzae No. of Time of onset of % Time of onset % survivor drug action Mortality of drug action Mortality (immobility time) (immobility time) 1 mg 10 - 0 1hr; 1P 0

5 mg 10 1hr; 1P 0 - 0

10 mg 10 1hr; 1D 10 3hr; 1D 10

25 mg 10 30min; 1P, 1hr; 1D 10 30min; 1P 0 50 mg 10 6hr; 1P & 1D 10 1hr; 1P & 1D 10

75 mg 10 - 0 3hr; 2P 0 100 mg 10 1hr; 1P, 24hr; 1D 10 6hr; 1D 10

Name of Insects % Mortality

+ ve Control Permethrin - ve Control (Coopex) at conc. 235.9 (solvent) µg/cm 2

Tribolium castaneum 100 0

Sitophilus oryzae 100 0

R = repellent, P = paralyzed, D= died

130

Table 69: Insecticide activity of R. fruticosus stem

Dose Tribolium castaneum Sitophilus oryzae No. of Time of onset of % Time of onset % survivor drug action Mortality of drug action Mortality (immobility time) (immobility time) 1 mg 10 2hr; 1P, 6hr; 1D 10 1hr; 1P 0 5 mg 10 1hr; 1P & 1D 10 3hr; 1D 10 10 mg 10 1hr; 1P, 24hr; 3D 30 1hr; 2P, 2hr; 1D 10 25 mg 10 10min; 1P, 2hr; 01D 10 1hr; 1P, 2hr; 2D 20 50 mg 10 30min; 1P, 6hr; 3D 10 1hr; 2 P 0 75 mg 10 2hr; 1D 10 30min; 1P,1D 10 100 mg 10 10min; 1P, 24hr; 4D 40 6hr; 2D 20 Name of Insects % Mortality

+ ve Control Permethrin - ve Control (Coopex) at conc. 235.9 (solvent) µg/cm 2 Tribolium castaneum 100 0

Sitophilus oryzae 100 0 R = repellent, P = paralyzed, D= died

Table 70: Insecticide activity of V. thapsus fruit

Dose Tribolium castaneum Sitophilus oryzae No. of Time of onset of % Time of onset % survivor drug action Mortality of drug action Mortality (immobility time) (immobility time) 1 mg 10 2hr; 1P 0 - 0

5 mg 10 - 0 - 0

10 mg 10 - 0 - 0

25 mg 10 - 0 3hr; 1P 0 50 mg 10 - 0 4hr; 1P 0 75 mg 10 1R 0 2R 0 100 mg 10 2R 0 2hr; 1D, 1R 10

Name of Insects % Mortality + ve Control Permethrin - ve Control (Copex) at conc. 235.9 (solvent) µg/cm 2

Tribolium castaneum 100 0

Sitophilus oryzae 100 0

R = repellent, P = paralyzed, D= died

131

Table 71: Insecticide activity of V. thapsus leaves

Dose Tribolium castaneum Sitophilus oryzae No. of Time of onset of drug % Time of onset of % survivor action (immobility Mortality drug action Mortality time) (immobility time) 1 mg 10 5min; 1P, 1hr; 1D 10 10min; 1P 0 5 mg 10 5min; 1P, 1hr; 2D 20 15min; 1P, 1hr; 1D 10 10 mg 10 5 min; 01P 10min; 1D 10 6hr; 2P 0 25 mg 10 - 0 1hr; 1D 10 50 mg 10 5min; 1P,12hr; 1D 10 1hr; 1P, 2hr; 1D 10 75 mg 10 2R 0 1hr; 1P, 1D, 2R 10 100 mg 10 10min; 1P 5min; 1D, 0 40min; 1D, 2R 10 3R Name of Insects % Mortality + ve Control Permethrin - ve Control (Copex) at conc. 235.9 (solvent) µg/cm 2 Tribolium castaneum 100 0 Sitophilus oryzae 100 0

R = repellent, P = paralyzed, D= died

Table 72: Insecticide activity of V. thapsus root

Dose Tribolium castaneum Sitophilus oryzae No. of Time of onset of drug % Time of onset of % survivor action (immobility Mortality drug action Mortality time) (immobility time) 1 mg 10 1hr; 1P, 2hr; 1D 10 2hr; 1P 0 5 mg 10 2hr; 1P 0 - 0 10 mg 10 5min; 1D, 6hr; 1P 10 2hr; 1D 10 25 mg 10 2hr; 1P, 6hr; 1D 10 2hr; 1P 0 50 mg 10 5min; 1P, 1hr; 1D 10 1hr; 1P 1hr; 1D 10 75 mg 10 1hr; 1P, 2hr; 1D, 3R 10 4hr; 2P, 5hr; 1D 10 100 mg 10 1hr; 1D, 2hr; 1P, 4R 10 2hr; 1D, 2R 10 Name of Insects % Mortality + ve Control Permethrin - ve Control (Copex) at conc. 235.9 (solvent) 2 µg/cm Tribolium castaneum 100 0 Sitophilus oryzae 100 0

R = repellent, P = paralyzed, D= died

132

Table 73: Insecticide activity of V. thapsus stem

Dose Tribolium castaneum Sitophilus oryzae No. of Time of onset of % Time of onset % survivor drug action Mortality of drug action Mortality (immobility time) (immobility time) 1 mg 10 - 0 - 0 5 mg 10 - 0 - 0 10 mg 10 - 10 - 0 25 mg 10 2hr; 01 D 10 2hr; 1P 0 50 mg 10 6hr; 3D 10 3hr; 2P 0 75 mg 10 - 10 50min; 1P, 1D 10 100 mg 10 24hr; 6R 0 60min; 1D 10 Name of Insects % Mortality

+ ve Control Permethrin - ve Control (Copex) at conc. 235.9 (solvent) µg/cm 2 Tribolium castaneum 100 0

Sitophilus oryzae 100 0 R = repellent, P = paralyzed, D= died

133

Table 74: Assessment of anti-helmintic activity of Rubus fruticosus fruit

Treatment Time (Doses ) 1 min 5min 10min 15min 30min 1hr 2hr 4hr 24hr mg/2ml Control N N N N N N N N N 5 mg 0++ 2 2 2 2 2 2 2 A 10 mg 0++ 2- 2-- 2- 3- 3- 3-- 4 D 25 mg 0 +++ 0++ 0 -- 3 3 4 D D 50 mg 0 +++ 0++ 0, 2-- 3-- 4-- 4-- - D 75 mg 0 +++ 0 +++ 2-- 3-- 3-- 4- 4- - D 100 mg 0 +++ 0 +++ 3-- 3-- 3--- 4- 4- - D

Number of replicates = three worms, Alive = A, Grade 0 = spontaneous motility, Grade 1 = Moderate motility, Grade 2 = less motility, Grade 3 = reduce touch evoke response, Grade 4 = total paralysis, Grade N = Normal, Grade D = Death, Secretions = S, Edema = E, Size reduction = ↓, Intensity = high+ moderate high ++, very high+++; low -, moderate low --, very low ---.

Doses Mean paralytic time Mean Death time 5mg Nil Alive 10mg Nil Alive 25mg 4hr 12hr 50mg 3hr 6hr 75mg 3hr 6hr 100mg 3hr 6hr

Comments: As we applied the extract we found abrupt increase in motility, then gradually reduction were observed, the drug effect was dose dependent, 5 and 10mg were the safe doses within 24 hours although 100mg paralyzed the worms within 3 hours.

134

Table 75: Assessment of anti-helmintic activity of Rubus fruticosus leaves

Treatment Time (Doses) 1 min 5min 10min 15min 30min 1hr 2hr 6hr 24hr mg/2ml Control N N N N N N N N N 5 mg 0+ 0+ 2 2 2 N - - A 10 mg 0++ 0+ 2 2 2 N - - A 25 mg 0++ 0++ 2-- 2--- 2- 3- 3-- A 50 mg 0 +++ 0 +++ 3- 2-- & 3 2- 3--- 3--- 4 D 75 mg 0 +++ 0 +++ 3-- 3 3++ ++ - 4 D 100 mg 0 +++ 0 +++ 3--, E 3-- +++, +++, - 4 D E

Number of replicates = three worms, Alive = A, Grade 0 = spontaneous motility, Grade 1 = Moderate motility, Grade 2 = less motility, Grade 3 = reduce touch evoke response, Grade 4 = total paralysis, Grade N = Normal, Grade D = Death, Secretions = S, Edema = E, Size reduction = ↓ , Intensity = high+ moderate high ++, very high+++; low -, moderate low --, very low ---.d= drops.

Doses Mean paralytic time Mean Death time 5 mg Nil Alive 10 mg Nil Alive 25 mg Nil Alive 50 mg 6Hrs 24hrs 75 mg 6Hrs 20hr 100 mg 5Hrs 18hr

Comments : With the application of drug hyperactivity and secretion were observed than gradually decrease in the motility. Here we found that even 25 mg was not toxic within 24 hours. No recovery was there after paralysis when placed in water. Dose dependent increase in toxicity was observed.

135

Table 76: Assessment of anti-helmintic activity of Rubus fruticosus root

Treatment Time (Doses) 1 min 5min 10min 15min 30min 1hr 2hr 4hr 24hr mg/2ml Control N N N N N N N N N 5 mg 0++ 0 N N N N N N A 10 mg 0 +++ 0 +++ 0 1 1 1,S 2 2 A 25 mg 0 +++ 0++ 0 2 2 3,S 4 4 4 50 mg 0 +++ 0++ 0, 2-- 2-- 3- 4 4 D 75 mg 0 +++ 1- 2-- 3-- 3-- 3-- 4 4-- D 100 mg 0 +++ 2-- 2-- 3-- 3 3-- 4 4-- D

Doses Mean paralytic time Mean Death time 5 mg Nil Alive 10 mg Nil Alive 25 mg 6hr Alive 50 mg 3hr 6hr 75 mg 2hr and 45min 6hr 100 mg 2hr & 30min 6hr

Comments : At 5, 10 and 25mg initially there was hyper motility and paralysis at 25 mg dosing no death was observed within 24 hours. Paralyses were observed at 25mg with in 6 hours. At 50, 75 and 100 mg they showed highly irritable behavior and death occur with in 6 hour.

136

Table 77: Assessment of anti-helmintic activity of Rubus fruticosus stem

Treatment Time (Doses) 1 min 5min 10min 15min 30min 1hr 2hr 4hr 24hr mg/2ml Control N N N N N N N N N 5 mg N 2 2 2 2 2 2 2 A 10 mg N 2- 2-- 2--- 3- 3- 3-- 3 A 25 mg 0 +++ 0++ 0 2 3 3 3 3 4 50 mg 0 +++ 0++ 0, 3 3 3 3 4 D 75 mg 0 +++ 1- 2-- 3-- 3 3 4 D - 100 mg 0 +++ 2 3 3 3 4 4 D -

Number of replicates = three worms, Alive = A, Grade 0 = spontaneous motility, Grade 1 = Moderate motility, Grade 2 = less motility, Grade 3 = reduce touch evoke response, Grade 4 = total paralysis, Grade N = Normal, Grade D = Death, Secretions = S, Edema = E, Si ze reduction = ↓ , Intensity = high+ moderate high ++, very high+++; low -, moderate low --, very low ---.

Doses Mean paralytic time Mean Death time 5 mg Nil Alive 10 mg Nil Alive 25 mg Nil Alive 50 mg 12-16hr 18-20 75 mg 2hr 3hr 100 mg 2hr 3hr

Comments : At 5mg, 10mg and 25mg initially there was hyper motility but later become normal. Paralyses were observed within 2-16 hours for higher doses and death from 3 to 20 hours.

137

Table 78: Assessment of anti-helmintic activity Verbascum thapsus fruit

Treatment Time (Doses) 1 min 5min 10min 15min 30min 1hr 2hr 4hr 24hr mg/2ml Control N N N N N N N N N 5 mg 0+ 2 2-- 3-- 4 D - - - 10 mg 0++ 2- 2-- 3-- 4 D - - - 25 mg 0 ++ 2-- 4 4 4 D - - - 50 mg 0 +++ 3-- 4 4 4 D - - - 75 mg 0 +++ 3- 4 4 4 D - - - 100 mg 0 +++ 4 4 4 4 D - - -

Number of replicates = three worms, Alive = A, Grade 0 = spontaneous motility, Grade 1 = Moderate motility, Grade 2 = less motility, Grade 3 = reduce touch evoke response, Grade 4 = total paralysis, Grade N = Normal, Grade D = Death, Secretions = S, Edema = E, Size reduction = ↓ , Intensity = high+ moderate high ++, very high+++; low -, moderate low --, very low ---

Doses Mean paralytic time Mean Death time 5 mg 30min 1hr 10 mg 30min 1hr 25 mg 10min 1hr 50 mg 10min 1hr 75 mg 10min. 1hr 100 mg 5min. 1hr

Comments : The hyper motility was observed even with small doses which were followed by decrease in motility then static mood, paralyses and death. The extract was so active that it killed all of the worms within 1 hour

138

Table 79: Assessment of anti-helmintic activity of Verbascum thapsus leaves

Treatment Time (Doses) 1 min 5min 10min 15min 30min 1hr 2hr 4hr 24hr mg/2ml Control N N N N N N N N N 5 mg 0++ 2 2 2 3 4 D - - 10 mg 0++ 2- 2-- 2--- 3- D - - - 25 mg 0 +++ 0++,S 3 4-- 3 D - - - 50 mg 0 +++ 0+3 4, 3-- 3-- D - - - 75 mg 0 ++++ 1-,S 4 3-- 4-- D - - - 100 mg 0 ++++ 3,S 4 4S 4S D, ↓ - - -

Number of replicates = three worms, Alive = A, Grade 0 = spontaneous motility, Grade 1 = Moderate motility, Grade 2 = less motility, Grade 3 = reduce touch evoke response, Grade 4 = total paralysis, Grade N = Normal, Grade D = Death, Secretions = S, Edema = E, Size reduction = ↓ , Intensity = high+ moderate high ++, very high+++; low -, moderate low --, very low ---.

Doses Mean paralytic time Mean Death time 5 mg 45min 75min 10 mg 45min 60min 25 mg 15min 45min 50 mg 10min 45min 75 mg 10min 40min 100 mg 10min 35min

Comments : Even at small concentration dose dependant hyper motility was present. The smallest dose i.e. 5mg caused paralysis in 45 minutes and death in 75 minutes. All dead worms had rigid body surfaces.

139

Table 80: Assessment of anti-helmintic activity of Verbascum thapsus root

Treatment Time (Doses) 1 min 5 min 10min 15min 30min 1hr 2hr 4hr 24hr 2mg/2ml Control N N N N N N N N N 5 mg 0++ 0 2 2 2 N N N A 10 mg 0++ 0 2 2 2 N N N A 25 mg 0 +++ 0+ 2 2 N N N N A 50 mg 0 +++ 0+ 0, 2 2 2-- 3 3-- D 75 mg 0 +++ 1- 2-- 2 2 3- 3- 3- D 100 mg 0 ++++ 2 2 2 2 3 3- 3- D

Number of replicates = three worms, Alive = A, Grade 0 = spontaneous motility, Grade 1 = Moderate motility, Grade 2 = less motility, Grade 3 = reduce touch evoke response, Grade 4 = total paralysis, Grade N = Normal, Grade D = Death, Secretions = S, Edem a = E, Size reduction = ↓, Intensity = high+ moderate high ++, very high+++; low -, moderate low --, very low ---.

Doses Mean paralytic Mean Death time time 5 mg Nil Alive 10 mg Nil Alive 25 mg Nil Alive 50 mg 10hr 24hr 75 mg 10hr 24hr 100 mg 8hr 24hrs

Comments : The drug was safe at 5, 10 and 25 mg although hyper motility was there when applied but the motility decreased with time. Motility was increased immediately as the dose of the drug increased however the drug had less toxicity to worms comparative to fruit or leaves of V. thapsus .

140

Table 81: Assessment of anti-helmintic activity of Verbascum thapsus stem

Treatment Time (Doses) 1 min 5min 10min 15min 30min 1hr 2hr 4hr 24hr mg/2ml Control N N N N N N N N N 5 mg N 2 2 2 2 2 2 3 A/S 10 mg 1 0 2 2 2 2 2 2- A/S 25 mg 0 0 2 2- 2--- 3 4 4- D 50 mg 0 0 0 3-- 3-- 3-- 4 4- D 75 mg 0 0 2 3-- 4 D - - - 100 mg 0 0 3 3, S 4, S D - - -

Number of replicates = three worms, Alive = A, Grade 0 = spontaneous motility, Grade 1 = Moderate motility, Grade 2 = less motility, Grade 3 = reduce touch evoke response, Grade 4 = total paralysis, Grade N = Normal, Grade D = Death, Secretions = S, Edema = E, Size reduction = ↓ , Intensity = high+ moderate high ++, very high+++; low -, moderate low --, very low ---.

Doses Mean paralytic time Mean Death time 5 mg Nil Alive 10 mg Nil Alive 25 mg 1hr &40 min 24hr 50 mg 1hr and 40min 24hr 75 mg 45min 60min 100 mg 45min 60min

Comments : This drug has more toxic effect in comparison with root of V. thapsus but we found it safe in low doses. Secretions were observed specially at low doses. The higher doses i.e. 75 and 100mg caused paralysis in 45 minutes followed by death in 60 minutes.

141

Table 82: Assessment of anti-helmintic activity of standard drug Niclosamide

Treatment Time (Doses) 1 min 5min 10min 15min 30min 1hr 2hr 4hr 24hr mg/2ml Control N N N N N N N N N 5 mg 0 2 2 3 4 D - - - 10 mg 0 2 2 3 4 D - - - 25 mg 0 2 2 3 4 D - - - 50 mg 0 +++ 2 2 3 4 D - - - 75 mg 0 ++ 2- 2-- 3 4 D - - - 100 mg 0 +++ 2 2 3 4 D - - -

Number of replicates = three worms, Alive = A, Grade 0 = spontaneous motility, Grade 1 = Moderate motility, Grade 2 = less motility, Grade 3 = reduce touch evoke response, Grade 4 = total paralysis, Grade N = Normal, Grade D = Death, Secretions = S, Edema = E, Size reduction = ↓ , Intensity = high+ moderate high ++, very high+++; low -, moderate low --, very low ---.

Doses Mean paralytic time Mean Death time 5 mg 35min 50min 10 mg 35min 45min 25 mg 35min 40min 50 mg 30min 40min 75 mg 30min 40min 100 mg 30min 40min

Comments : The standard drug was applied in the same doses as crude extract we found it very toxic. Motility was increased immediately as the dose of the drug increased. Paralysis was observed within 35 minutes and death within 50 minutes even at small applied dose.

142

Table 83: Brine shrimp toxicity of Rubus fruticosus fruit

Probit analysis (Finney method) log normal distribution at α= 0.05

Log10 dose Actual % Probit % N R Chi-square stimulus 1 0.1 0.0418 10 1 0.8096 2 0.3 0.4624 10 3 0.5701 3 0.975 0.9383 10 9.75 0.0143 LD 50 114.2281 µg/ml Log10(LD 50 ) 2.0578

Table 84: Brine shrimp toxicity of Rubus fruticosus leaves

Probit analysis (Finney method) log normal distribution at α= 0.05

Log10 dose Actual % Probit % N R Chi-square stimulus 1 0.3 0.2261 10 3 0.2415 2 0.5 0.6557 10 5 0.3699 3 0.975 0.9398 10 9.75 0.0131 LD 50 44.89 µg/ml Log10(LD 50 ) 1.6522

Table 85: Brine shrimp toxicity of Rubus fruticosus roots

Probit analysis (Finney method) log normal distribution at α= 0.05

Log10 dose Actual % Probit % N R Chi-square stimulus 1 0.3 0.2981 10 3 0.0001 2 0.9 0.9031 10 9 0.0001 3 0.975 0.999 10 9.75 0.0058 LD 50 19.4803 µg/ml Log10(LD 50 ) 1.2896

143

Table 86: Brine shrimp toxicity of Rubus fruticosus stem

Probit analysis (Finney method) log normal distribution at α= 0.05

Log10 dose Actual % Probit % N R Chi-square stimulus 1 0.2 0.1081 10 2 0.7818 2 0.3 0.5218 10 3 0.9426 3 0.975 0.9109 10 9.75 0.0452 LD 50 90.7237 µg/ml Log10(LD 50 ) 1.9577

Table 87: Brine shrimp toxicity of Verbascum thapsus fruit

Probit analysis (Finney method) log normal distribution at α= 0.05

Log10 dose Actual % Probit % N R Chi-square stimulus 1 0.7 0.6855 10 7 0.0031 2 0.9 0.9235 10 9 0.006 3 0.975 0.9912 10 9.75 0.0027 LD 50 3.0872 µg/ml Log10(LD 50 ) 0.4896

Table 88: Brine shrimp toxicity of Verbascum thapsus leaves

Probit analysis (Fin ney method) log normal distribution at α= 0.05

Log10 dose Actual % Probit % N R Chi-square stimulus 1 0.9 0.9 10 9 0 2 0.975 1 10 9.75 0.0062 3 0.975 1 10 9.75 0.0062 LD 50 3.4207 µg/ml Log10(LD 50 ) 0.5341

144

Table 89: Brine shrimp toxicity of Verbascum thapsus root extract

Probit analysis (Finney method) log normal distribution at α= 0.05

Log10 dose Actual % Probit % N R Chi-square stimulus 1 0.2 0.1729 10 2 0.0424 2 0.4 0.4606 10 4 0.0797 3 0.8 0.7718 10 8 0.0103 LD 50 130.9955 µg/ml Log10(LD 50 ) 2.1173

Table 90: Brine shrimp toxicity of Verbascum thapsus stem extract

Probit analysis (Finney method) log normal distribution at α= 0.05

Log10 dose Actual % Probit % N R Chi- stimulus square 1 0.3 0.3636 10 3 0.1112 2 0.7 0.5713 10 7 0.2902 3 0.7 0.7605 10 7 0.0482 LD 50 45.728 µg/ml Log10(LD 50 ) 1.6602

Table 91: Brine shrimp toxicity of Cyclophosphamide (Standard)

Probit analysis (Finney method) log normal distribution at α= 0.05

Log10 dose Actual % Probit % N R Chi- stimulus square 1 0.1 0.1351 10 1 0.0909 2 0.3 0.2243 10 3 0.0255 3 0.3 0.3399 10 3 0.0468 LD 50 15.708 µg/ml Log10(LD 50 ) 4.196

Actual Percent - the ratio of the count to the sample size (R/N). Probit Percent - the estimated ratio (R/N) based on the probit model. N - the sample size. R - the count (number responding). Chi-Square - the Chi-Square statistic for testing the significance (non-zero) of the difference

145

Fig.1 : Thin layer Chromtography in Visible, UV 254nm and UV366

146

Fig.2 : Thin layer Chromtography in Visible, UV 254nm and UV366

147

DPPH assay of R. fruticosus

100 90 80 70 60 50 RF Fruit 40 30 RF leaves 20 RF root % Radical sacvenging Radical % 10 0 RF stem

Conc. of sample mg/ml

DPPH assay of V. thapsus

100 90 80 70 60 50 VT fruit 40 30 VT leaves 20 VT root % Radical scavenging Radical % 10 0 VT stem

Conc. of sample mg/ml

Graph 1: Antioxidant activity of R. fruticosus and V. thapsus 148

DPPH assay of Ascorbic acid

100 80 60 40 20

% Radical scavenging Radical% 0

Conc. of Ascorbic acid mg/ml

ABTS assay of R. fruticosus 120 100 80 60 40 % inhibition % 50µg/ml 20 100µg/ml 0

Samples

Graph 1: Antioxidant activity of R. fruticosus and V. thapsus

149

ABTS assay of V. thapsus 120 100 80 60 40 % inhibition % 50µg/ml 20 100µg/ml 0

Samples

Nitric oxide assay of R. fruticosus 100 90 80 70 60 50 40

% inhibition % 30 50µg/ml 20 10 100µg/ml 0

Samples

Graph 1: Antioxidant activity of R. fruticosus and V. thapsus

150

Nitric oxide assay of V. thapsus 120 100 80 60 40 % inhibition % 50µg/ml 20 100µg/ml 0

Samples

Graph 1: Antioxidant activity of R. fruticosus and V. thapsus

151

R. fruticosus (fruit) 80 70 60 50 40 30 1st phase % inhibition % 20 2nd phase 10 0 100 300 500 Aspirin CS 50 CS 100 CS 150 Dose as mg/kg

R. fruticosus (leaves) 80 70 60 50 40 30 1st phase % inhibition % 20 2nd phase 10 0 100 300 500 Aspirin CS 50 CS 100 CS 150 Dose as mg/kg

Graph 2: Anti-inflammatory activity (Formalin induced inflammation)

152

R. fruticosus (root) 80 70 60 50 40 30 1st phase % inhibition % 20 2nd phase 10 0 100 300 500 Aspirin CS 50 CS 100 CS 150 Dose as mg/kg

R. fruticosus (stem) 80 70 60 50 40 30 1st phase % inhibition % 20 2nd phase 10 0 100 300 500 Aspirin CS 50 CS 100 CS 150 Dose as mg/kg

Graph 2: Anti-inflammatory activity (Formalin induced inflammation)

153

V. thapsus (fruit) 80 70 60 50 40 30 1st phase % inhibition % 20 2nd phase 10 0 100 300 500 Aspirin CS 50 CS 100 CS 150 Dose as mg/kg

V. thapsus (leaves) 80 70 60 50 40 30 1st phase % inhibition % 20 2nd phase 10 0 100 300 500 Aspirin CS 50 CS 100 CS 150 Dose as mg/kg

Graph 2: Anti-inflammatory activity (Formalin induced inflammation)

154

V. thapsus (root) 80 70 60 50 40 30 1st phase % inhibition % 20 2nd phase 10 0 100 300 500 Aspirin CS 50 CS 100 CS 150 Dose as mg/kg

V. thapsus (stem) 80 70 60 50 40 30 1st phase % inhibition % 20 2nd phase 10 0 100 300 500 Aspirin CS 50 CS 100 CS 150 Dose as mg/kg

Graph 2: Anti-inflammatory activity (Formalin induced inflammation)

155

R. fruticosus (fruit)

45 40 35 30 25 1st hour 20 15 2nd hour % inhibition % 10 3rd hour 5 0 100 300 500 Aspirin(300) Dose as mg/kg

R. fruticosus (leaves)

45 40 35 30 25 1st hour 20 15 2nd hour % inhibition % 10 3rd hour 5 0 100 300 500 Aspirin(300) Dose as mg/kg

Graph 3: Anti-inflammatory activity (Carrageenan induced inflammation)

156

R. fruticosus (root)

35 30 25 20 1st hour 15 2nd hour % inhibition % 10 3rd hour 5 0 100 300 500 Aspirin(300) Dose as mg/kg

R. fruticosus (stem)

35 30 25 20 1st hour 15 2nd hour % inhibition % 10 3rd hour 5 0 100 300 500 Aspirin(300) Dose as mg/kg

Graph 3: Anti-inflammatory activity (Carrageenan induced inflammation)

157

V. thapsus (fruit)

40 35 30 25 20 1st hour 15 2nd hour % inhibition % 10 3rd hour 5 0 100 300 500 Aspirin(300) Dose as mg/kg

V. thapsus (leaves)

40 35 30 25 20 1st hour 15 2nd hour % inhibition % 10 3rd hour 5 0 100 300 500 Aspirin(300) Dose as mg/kg

Graph 3: Anti-inflammatory activity (Carrageenan induced inflammation)

158

V. thapsus (root)

35 30 25 20 1st hour 15 2nd hour % inhibition % 10 3rd hour 5 0 100 300 500 Aspirin(300) Dose as mg/kg

V. thapsus (stem)

35 30 25 20 1st hour 15 2nd hour % inhibition % 10 3rd hour 5 0 100 300 500 Aspirin(300) Dose as mg/kg

Graph 3: Anti-inflammatory activity (Carrageenan induced inflammation)

159

R. fruticosus (fruit) 50 45 40 35 30 Control 25 100mg/kg 20 15 300mg/kg 10 500mg/kg

Response time in seconds in time Response 5 Aspirin(300mg/kg) 0 1 2 3 4 5 6 7 8 9 10 Time in hours

R. fruticosus (leaves) 50 45 40 35 30 Control 25 100mg/kg 20 15 300mg/kg 10 500mg/kg

Response time in seconds in time Response 5 Aspirin(300mg/kg) 0 1 2 3 4 5 6 7 8 9 10 Time in hours

Graph 4: The effect of hot plate activity

160

R. fruticosus (root) 50 45 40 35 30 Control 25 100mg/kg 20 15 300mg/kg 10 500mg/kg

Response time in seconds in time Response 5 Aspirin(300mg/kg) 0 1 2 3 4 5 6 7 8 9 10 Time in hours

R. fruticosus (stem) 50 45 40 35 30 Control 25 100mg/kg 20 15 300mg/kg 10 500mg/kg

Response time in seconds in time Response 5 Aspirin(300mg/kg) 0 1 2 3 4 5 6 7 8 9 10 Time in hours

Graph 4: The effect of hot plate activity

161

V. thapsus (fruit) 50 45 40 35 30 Control 25 100mg/kg 20 15 300mg/kg 10 500mg/kg

Response time in seconds in time Response 5 Aspirin(300mg/kg) 0 1 2 3 4 5 6 7 8 9 10 Time in hours

V. thapsus (leaves) 50 45 40 35 30 Control 25 100mg/kg 20 15 300mg/kg 10 500mg/kg

Response time in seconds in time Response 5 Aspirin(300mg/kg) 0 1 2 3 4 5 6 7 8 9 10 Time in hours

Graph 4: The effect of hot plate activity

162

V. thapsus (root) 50 45 40 35 30 Control 25 100mg/kg 20 15 300mg/kg 10 500mg/kg

Response time in seconds in time Response 5 Aspirin(300mg/kg) 0 1 2 3 4 5 6 7 8 9 10 Time in hours

V. thapsus (stem) 50 45 40 35 30 Control 25 100mg/kg 20 15 300mg/kg 10 500mg/kg

Response time in seconds in time Response 5 Aspirin(300mg/kg) 0 1 2 3 4 5 6 7 8 9 10 Time in hours

Graph 4: The effect of hot plate activity

163

R. fruticosus (fruit)

70 60 50 40 30 1st phase

% inhibition % 20 2nd phase 10 0 100 300 500 Aspirin Dose as mg/kg

R. fruticosus (leaves)

80 70 60 50 40 1st phase 30 % inhibition % 20 2nd phase 10 0 100 300 500 Aspirin Dose as mg/kg

Graph 5: The results of analgesic activity by acetic acid induced writhing test 164

R. fruticosus (root)

70 60 50 40 30 1st phase

% inhibition % 20 2nd phase 10 0 100 300 500 Aspirin Dose as mg/kg

R. fruticosus (stem)

60 50 40 30 1st phase 20 % inhibition % 2nd phase 10 0 100 300 500 Aspirin Dose as mg/kg

Graph 5: The results of analgesic activity by acetic acid induced writhing test 165

V. thapsus (fruit)

80 70 60 50 40 1st phase 30 % inhibition % 20 2nd phase 10 0 100 300 500 Aspirin Dose as mg/kg

V. thapsus (leaves)

70 60 50 40 30 1st phase

% inhibition % 20 2nd phase 10 0 100 300 500 Aspirin Dose as mg/kg

Graph 5: The results of analgesic activity by acetic acid induced writhing test 166

V. thapsus (root)

60 50 40 30 1st phase 20 % inhibition % 2nd phase 10 0 100 300 500 Aspirin Dose as mg/kg

V. thapsus (stem)

60 50 40 30 1st phase 20 % inhibition % 2nd phase 10 0 100 300 500 Aspirin Dose as mg/kg

Graph 5: The results of analgesic activity by acetic acid induced writhing test

167

R. fruticosus (fruit) 8 7 6

5 Control(0.5ml saline) 4 100mg/kg 3 300mg/kg 2 500mg/kg

Latency time in seconds in time Latency 1 Aspirin(300mg/kg) 0 1 2 3 4 5 6 7 8 9 10 Time in hours

R. fruticosus (leaves) 7 6 5 4 Control(0.5ml saline) 3 100mg/kg 300mg/kg 2 500mg/kg 1 Latency time in seconds in time Latency Aspirin(300mg/kg) 0 1 2 3 4 5 6 7 8 9 10 Time in hours

Graph 6: Analgesic activity of drugs through tail flick water bath

168

R. fruticosus (root) 7 6 5 4 Control(0.5ml saline) 3 100mg/kg 300mg/kg 2 500mg/kg

Latency time in seconds in time Latency 1 Aspirin(300mg/kg) 0 1 2 3 4 5 6 7 8 9 10 Time in hours

R. fruticosus (stem) 7 6 5 4 Control(0.5ml saline) 3 100mg/kg 300mg/kg 2 500mg/kg 1 Latency time in seconds in time Latency Aspirin(300mg/kg) 0 1 2 3 4 5 6 7 8 9 10 Time in hours

Graph 6: Analgesic activity of drugs through tail flick water bath

169

V. thapsus (fruit) 7 6 5 4 Control(0.5ml saline) 3 100mg/kg 300mg/kg 2 500mg/kg

Latency time in seconds in time Latency 1 Aspirin(300mg/kg) 0 1 2 3 4 5 6 7 8 9 10 Time in hours

V. thapsus (leaves) 7 6 5 4 Control(0.5ml saline) 3 100mg/kg 300mg/kg 2 500mg/kg 1 Latency time in seconds in time Latency Aspirin(300mg/kg) 0 1 2 3 4 5 6 7 8 9 10 Time in hours

Graph 6: Analgesic activity of drugs through tail flick water bath

170

V. thapsus (root) 7 6 5 4 Control(0.5ml saline) 3 100mg/kg 300mg/kg 2 500mg/kg

Latency time in seconds in time Latency 1 Aspirin(300mg/kg) 0 1 2 3 4 5 6 7 8 9 10 Time in hours

V. thapsus (stem) 7 6 5 4 Control(0.5ml saline) 3 100mg/kg 300mg/kg 2 500mg/kg 1 Latency time in seconds in time Latency Aspirin(300mg/kg) 0 1 2 3 4 5 6 7 8 9 10 Time in hours

Graph 6: Analgesic activity of drugs through tail flick water bath

171

R. fruticosus (fruit) 600 500 400 300 200 100 Open field Number of counts of Number 0 Head dip

Dose as mg/kg

R. fruticosus (leaves) 350 300 250 200 150 100 50 Open field Number of counts of Number 0 Head dip

Dose as mg/kg

Graph 7: The effect of open field and head dip activity 172

R. fruticosus (root) 450 400 350 300 250 200 150 100 Open field

Number of counts of Number 50 0 Head dip

Dose as mg/kg

R. fruticosus (stem) 350 300 250 200 150 100 50 Open field Number of counts of Number 0 Head dip

Dose as mg/kg

Graph 7: The effect of open field and head dip activity 173

V. thapsus (fruit) 400 350 300 250 200 150 100 50 Open field Number of counts of Number 0 Head dip

Dose as mg/kg

V. thapsus (leaves) 350 300 250 200 150 100 50 Open field Number of counts of Number 0 Head dip

Dose as mg/kg

Graph 7: The effect of open field and head dip activity 174

V. thapsus (root) 600 500 400 300 200 100 Open field Number of counts of Number 0 Head dip

Dose as mg/kg

V. thapsus (stem) 600 500 400 300 200 100 Open field Number of counts of Number 0 Head dip

Dose as mg/kg

Graph 7: The effect of open field and head dip activity 175

R. fruticosus (fruit) 100 90 80 70 60 50 40

Response 30 20 10 Cage cross 0 Rearing

Dose as mg/kg

R. fruticosus (leaves) 90 80 70 60 50 40 30 Response 20 10 Cage cross 0 Rearing

Dose as mg/kg

Graph 8: The effect of cage cross and rearing activity 176

R. fruticosus (root) 90 80 70 60 50 40 30 Response 20 10 Cage cross 0 Rearing

Dose as mg/kg

R. fruticosus (stem) 90 80 70 60 50 40 30 Response 20 10 Cage cross 0 Rearing

Dose as mg/kg

Graph 8: The effect of cage cross and rearing activity 177

V. thapsus (fruit) 90 80 70 60 50 40 30 Response 20 10 Cage cross 0 Rearing

Dose as mg/kg

V. thapsus (leaves) 90 80 70 60 50 40 30 Response 20 10 Cage cross 0 Rearing

Dose as mg/kg

Graph 8: The effect of cage cross and rearing activity

178

V. thapsus (root) 90 80 70 60 50 40 30 Response 20 10 Cage cross 0 Rearing

Dose as mg/kg

V. thapsus (stem) 90 80 70 60 50 40 30 Response 20 10 Cage cross 0 Rearing

Dose as mg/kg

Graph 8: The effect of cage cross and rearing activity 179

R. fruticosus (fruit) 6 5 4 3 2 Response 1 Mobility time 0 Immobility time

Dose as mg/kg

R. fruticosus (leaves) 4.5 4 3.5 3 2.5 2 1.5 Response 1 0.5 Mobility time 0 Immobility time

Dose as mg/kg

Graph 9: Forced swimming test (The effect of drug on mobility time) 180

R. fruticosus (root) 5 4.5 4 3.5 3 2.5 2

Response 1.5 1 0.5 Mobility time 0 Immobility time

Dose as mg/kg

R. fruticosus (stem) 4.5 4 3.5 3 2.5 2 1.5 Response 1 0.5 Mobility time 0 Immobility time

Dose as mg/kg

Graph 9: Forced swimming test (The effect of drug on mobility time) 181

V. thapsus (fruit) 4.5 4 3.5 3 2.5 2 1.5 Response 1 0.5 Mobility time 0 Immobility time

Dose as mg/kg

V. thapsus (leaves) 6 5 4 3 2 Response 1 Mobility time 0 Immobility time

Dose as mg/kg

Graph 9: Forced swimming test (The effect of drug on mobility time) 182

V. thapsus (root) 4.5 4 3.5 3 2.5 2 1.5 Response 1 0.5 Mobility time 0 Immobility time

Dose as mg/kg

V. thapsus (stem) 4.5 4 3.5 3 2.5 2 1.5 Response 1 0.5 Mobility time 0 Immobility time

Dose as mg/kg

Graph 9: Forced swimming test (The effect of drug on mobility time)

183

R. fruticosus (fruit) E.coli 30

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

R. fruticosus (fruit ) S.typhi 25

5 Zone of inhibition in mm in inhibition of Zone 0

Dose µg/disc

Graph 10: Antibacterial activity against different microorganisms

184

R. fruticosus (fruit) S.aureus 25

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

R. fruticosus (fruit) P.mirabilis 25

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

Graph 10: Antibacterial activity against different microorganisms

185

R. fruticosus (fruit) M.luteus 25

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

R. fruticosus (fruit) Citrobacter 20 18 16 14 12 10 8 6 4 Zone of inhibition in mm in inhibition of Zone 2 0

Dose µg/disc

Graph 10: Antibacterial activity against different microorganisms

186

R.fruticosus (fruit) B.subtilis 25

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

R. fruticosus (fruit) P.aeruginosa 25

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

Graph 10: Antibacterial activity against different microorganisms

187

R. fruticosus (leaves) E.coli 30

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

R. fruticosus (leaves) S.typhi 25

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

Graph 10: Antibacterial activity against different microorganisms

188

R. fruticosus (leaves) S.aureus 25

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

R. fruticosus (leaves) P.mirabilis 25

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

Graph 10: Antibacterial activity against different microorganisms

189

R. fruticosus (leaves) M.luteus 25

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

R. fruticosus (leaves) Citrobacter 20 18 16 14 12 10 8 6 4 Zone of inhibition in mm in inhibition of Zone 2 0

Dose µg/disc

Graph 10: Antibacterial activity against different microorganisms

190

R. fruticosus (leaves) B.subtilis 25

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

R. fruticosus (leaves) P.aeruginosa 25

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

Graph 10: Antibacterial activity against different microorganisms

191

R. fruticosus (root) E.coli 30

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

R. fruticosus (root) S.typhi 25

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

Graph 10: Antibacterial activity against different microorganisms

192

R. fruticosus (root) S.aureus 25

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

R. fruticosus (root) P.mirabilis 25

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

Graph 10: Antibacterial activity against different microorganisms

193

R. fruticosus (root) M.luteus 25

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

R. fruticosus (root) Citrobacter 20 18 16 14 12 10 8 6 4 Zone of inhibition in mm in inhibition of Zone 2 0

Dose µg/disc

Graph 10: Antibacterial activity against different microorganisms

194

R. fruticosus (root) B.subtilis 25

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

R. fruticosus (root) P.aeruginosa 25

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

Graph 10: Antibacterial activity against different microorganisms

195

R. fruticosus (stem) E.coli 30

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

R. fruticosus (stem) S.typhi 25

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

Graph 10: Antibacterial activity against different microorganisms

196

R. fruticosus (stem) S.aureus 25

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

R. fruticosus (stem) P.mirabilis 25

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

Graph 10: Antibacterial activity against different microorganisms

197

R. fruticosus (stem) M.luteus 25

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

R. fruticosus (stem) Citrobacter 20 18 16 14 12 10 8 6 4 Zone of inhibition in mm in inhibition of Zone 2 0

Dose µg/disc

Graph 10: Antibacterial activity against different microorganisms

198

R. fruticosus (stem) B.subtilis 25

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

R. fruticosus (stem) P.aeruginosa 25

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

Graph 10: Antibacterial activity against different microorganisms

199

V. thapsus (fruit) E.coli 30

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

V. thapsus (fruit) S.typhi 25

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

Graph 10: Antibacterial activity against different microorganisms

200

V. thapsus (fruit) S.aureus 25

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

V. thapsus (fruit) P.mirabilis 25

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

Graph 10: Antibacterial activity against different microorganisms

201

V. thapsus (fruit) M.luteus 25

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

V. thapsus (fruit) Citrobacter 20 18 16 14 12 10 8 6 4 Zone of inhibition in mm in inhibition of Zone 2 0

Dose µg/disc

Graph 10: Antibacterial activity against different microorganisms

202

V. thapsus (fruit) B.subtilis 25

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

V. thapsus (fruit) P.aeruginosa 25

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

Graph 10: Antibacterial activity against different microorganisms

203

V. thapsus (leaves) E.coli 30

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

V. thapsus (leaves) S.typhi 25

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

Graph 10: Antibacterial activity against different microorganisms

204

V. thapsus (leaves) S.aureus 25

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

V. thapsus (leaves) P.mirabilis

5 Zone of inhibition in mm in inhibition of Zone 0

Dose µg/disc

Graph 10: Antibacterial activity against different microorganisms

205

V. thapsus (leaves) M.luteus 25

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

V. thapsus (leaves) Citrobacter 20 18 16 14 12 10 8 6 4 Zone of inhibition in mm in inhibition of Zone 2 0

Dose µg/disc

Graph 10: Antibacterial activity against different microorganisms

206

V. thapsus (leaves) B.subtilis 25

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

V. thapsus (leaves) P.aeruginosa 25

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

Graph 10: Antibacterial activity against different microorganisms

207

V. thapsus (root) E.coli 30

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

V. thapsus (root) S.typhi 25

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

Graph 10: Antibacterial activity against different microorganisms

208

V. thapsus (root) S.aureus 25

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

V. thapsus (root) P.mirabilis 25

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

Graph 10: Antibacterial activity against different microorganisms

209

V. thapsus (root) M.luteus 25

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

V. thapsus (root) Citrobacter 20 18 16 14 12 10 8 6 4 Zone of inhibition in mm in inhibition of Zone 2 0

Dose µg/disc

Graph 10: Antibacterial activity against different microorganisms

210

V. thapsus (root) B.subtilis 25

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

V. thapsus (root) P.aeruginosa 25

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

Graph 10: Antibacterial activity against different microorganisms

211

V. thapsus (stem) E.coli 30

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

V. thapsus (stem) S.typhi 25

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

Graph 10: Antibacterial activity against different microorganisms

212

V. thapsus (stem) S.aureus 25

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

V. thapsus (stem) P.mirabilis 25

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

Graph 10: Antibacterial activity against different microorganisms

213

V. thapsus (stem) M.luteus 25

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

V. thapsus (stem) Citrobacter 20 18 16 14 12 10 8 6 4 Zone of inhibition in mm in inhibition of Zone 2 0

Dose µg/disc

Graph 10: Antibacterial activity against different microorganisms

214

V. thapsus (stem) B.subtilis 25

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

V. thapsus (stem) P.aeruginosa 25

5 Zone of inhibition in mm in inhibition of Zone

Dose µg/disc

Graph 10: Antibacterial activity against different microorganisms

215

Graph 11: Brine shrimp lethality test of methanolic extracts

216

Graph 11: Brine shrimp lethality test of methanolic extracts

217

Graph 11: Brine shrimp lethality test of methanolic extracts

218

Graph 11: Brine shrimp lethality test of methanolic extracts

219

Graph 11: Brine shrimp lethality test of methanolic extracts

220

DISCUSSION

The aim of exploring the hidden potential of the two plants Rubus fruticosus L. and Verbascum thapsus L. is based on the traditional importance in local community of district Dir (KPK). The investigations are presented and discussed in the following manner to understand the worth of these plants in light of our research findings.

1. Pharmacognostic studies a) R. fruticosus Four methanolic extracts of R. fruticosus were screened through various chemical reagents for different groups of compounds. These qualitative colourometric methods provide base for advance studies. We found our tests positive for the presence of aromatic ring based compounds, polyphenolic compounds, carbohydrates, tannins and flavonoids (Table 1a and 1b). All four extracts of R. fruticosus (RFF, RFL, RFR and RFS) were determined to be free of cyanide.

Fluorescence analysis of powder of RFL, RFR and RFS were treated with different reagents or solvents and their observed characteristics colours were recorded (Table 3-5). Similarly the fluorescence behaviour of RFF, RFL, RFR and RFF in different solvents in normal, UV 254nm and UV 366nm were studied (Table 9). These analyses provide standards and information about identity, authenticity and quality of R. fruticosus for future investigations or uses.

Thin layer chromatography of RFF, RFL, RFR and RFS was performed in two solvent systems (Table 11, Fig.1). The TLC plates were observed under UV light at 254 nm and 366 nm. The extracts showed characteristic bands. The distances of these spots/bands were measured. Rf values for each spot were calculated and recorded. This TLC information provides help in marker approach regarding quality and identification of R. fruticosus plant materials.

221 b) V. thapsus The four types of methanolic extracts from V. thapsus i.e. fruit (VTF), leaves (VTL), root (VTR) and stem (VTS) were screened as follows.

The presence of different chemical constituents in V. thapsus was identified by reactions with different chemical reagents described in the experimental section. These reactions showed us characteristics colours which indicate the presence or absence of various groups of compounds. We found indication for aromatic ringed compounds, polyphenols, alkaloids, flavonoids and aminosalicylates in our samples (Table 2a and 2b).

Powder drug studies of V. thapsus with different solvents or reagents are given in Table 6 for VTL, Table 7 for VTR and Table 8 for VTS. These powders were observed in day light, UV 254 nm and UV 366 nm.

2. Antioxidant studies Reactive oxygen species and free radicals are produced within the living animals during metabolism. These reactive species interact with bio-molecules and results cell or tissue damage (Yen and Chen, 1995). However the consumption of natural antioxidants usually of plant origin reduces these reactive species and thus prevents injuries in living cells or tissues. So antioxidant potential various parts of both species was determined by battery of assays including DPPH, ABTS and Nitric oxide free radical assay methods.

222 a) R. fruticosus 10 dilutions of methanolic extracts were made and assayed through photometric method using DPPH assay method (Table 13, Graph 1). However 50 µg/ml and 100 µg/ml of all four samples along with standard Ascorbic acid were used in ABTS (Table 15, Graph 1) and Nitric oxide assay method (Table 16, Graph 1). We found that RFF had the highest % of radical scavenging then RFL followed by RFR and then RFS. We found the same trend of antioxidant potentials in all three applied methods.

Literature study revealed that antioxidant activity may be due to anthocyanin (Talavera et al ., 2005; Fan- Chiang and Wrolstad, 2005; Wu and Prior, 2005; Elisia and David, 2008; Ogawa et al ., 2008) and carotenoids (Marinova and Ribarova, 2007) especially in fruit. The role of flavonoids (Gudej and Tomczyk, 2004; Sanjust et al ., 2008) and tannins (Nakahara et al ., 1996; Carlsen et al ., 2003) in antioxidant activity can’t be neglected. We found fruit the most potent antioxidant comparatively. We found the antioxidant capacity in the following order RFF > RFL > RFR > RFS. b) V. thapsus 10 concentrations were made and assayed through photometric method using DPPH assay method (Table 14, Graph 1). However 50 µg/ml and 100 µg/ml of all four samples along with standard Ascorbic acid were used in ABTS (Table 15, Graph 1) and Nitric oxide assay method (Table 16, Graph 1). In our studies we found that VTR had maximum antioxidant capacity and then VTF.

Literature study showed that antioxidant activity may be due to the presence of phenolic acids and flavonoids (Katalinic et al ., 2006; Antal, 2010). Verbascoside was also found to have nitric oxide radical scavenging activity (Kimura et al ., 1987; Panchal et al ., 2010) so may have a strong contribution towards antioxidant capability of V. thapsus.

We found strong free radical scavenging capacity in both plants so we may use these plants in the treatment of chronic illnesses.

223

3. Anti-inflammatory activity Inflammatory mechanism involves activation of signaling pathways in regular succession without gaps leading to the production of both pro- and anti-inflammatory mediators. However pro-inflammatory mediators are focused during anti-inflammatory action because they initiate inflammation (Lawrence et al ., 2001).

Two pathways are followed during inflammation. Histamines, serotonin and bradykinin are released in first phase while prostaglandins are released in second phase (Vyas et al., 2008). Carrageenan and formalin injection into the rat or mice paw provokes a local acute inflammatory reaction (Rezazadeh et al., 2005). Currently various synthetic NSAIDs are available to treat various inflammatory disorders but they are not free from side effects so a part of continuous investigations in our group for natural anti- inflammatory agent we carried out the following anti-inflammatory activities of the selected two plants. a) R. fruticosus In present work R. fruticosus various parts at 100, 300 and 500 mg/kg per oral dose were evaluated for the anti-inflammatory action using formalin (Table 17, Graph 2) and rat paw oedema test (Table 18, Graph 3). Aspirin was used as standard drug. Significant reduction in inflammation was observed. RFF showed significant inhibition but almost same in both phases. The significant dose dependent inhibitions were observed at a dose of 500 mg/kg, for first and second phase for RFL.

Analgesics and anti-inflammatory drugs behave in different ways in formalin biphasic response test i.e. in first phase and second phase of inflammation (Tjolsen et al ., 1992). RFF was observed for maximum activity in second phase. RFL showed maximum response in first phase compared to the rest of R. fruticosus extracts applied. RFS showed the least inhibitions at 500 mg/kg for first and second phase, respectively. The % inhibition of RFF and RFL at a dose of 500 mg was almost comparable with aspirin in first phase.

224

In case of carrageenan induced inflammation the crude extract of R. fruticosus showed dose dependant increase in the percentage of inhibition of edema which remained for 3 hour. The results were measured in mm and percent inhibition were calculated for four parts (RFF, RFL, RFF and RFS) of R. fruticosus . Aspirin, as a standard at dose of 300 mg/kg markedly reduced the edema hourly observed. The highest percent of inhibition with rat paw edema test were observed with R. fruticosus leaves and fruit extracts at 500 mg/kg.

Similar results were obtained in RFF, RFL and RFR except there is a change in intensity. RFL was slightly more effective than RFF. Among all crude extract RFL was found to be more effective as anti-inflammatory agent. b) V. thapsus The results of formalin induced inflammation illustrated in Table 17 and Graph 2 showed that extracts of V. thapsus (100, 300 and 500 mg/kg) caused a dose dependent inhibition. There were significant inhibitions observed at p≤0.05 at a dose of 500 mg/kg, for first and second phase for VTF. VTL also showed significant inhibitions at 500 mg/kg. However VTR and VTS did not show significant inhibitions at 500 mg/kg for first and second phase, respectively.

The inhibitory activity of V. thapsus extracts against Carrageenan induced paw edema was presented in Table 18 and Graph 3. The results were measured in mm and percent inhibition were calculated for four parts (VTF, VTL, VTF and VTS) of V. thapsus . Aspirin, as a standard at dose of 300 mg/kg markedly reduced the edema.

It is believed that acteoside (Verbascoside) and aucubin (Groeger et al ., 1967; Seifert et al ., 1985) may be responsible for antinociceptive and anti-inflammatory effects (Pascual Teresa et al ., 1978; Kupeli et al ., 2007; Speranza et al ., 2010).

225

4. Analgesic activity Analgesia or pain has been defined by the International Association for the Study of Pain “an unpleasant sensory and emotional experience associated with actual or potential tissue damage” (Hartrick, 2007; Sinatra et al ., 2010). Pain is a physiological complex process that is usually produced through the activation of nociceptors by thermal, mechanical or chemical tissue injuries. Nociceptors are peripheral endings of A-delta and C sensory fibers that has major role in pain detection (Sinatra et al ., 2010). We used temperature induced (hot plate and tail flick) and chemically induced (writhing test) pain models in our studies to evaluate the analgesic effect of R. fruticosus and V. thapsus . a) R. fruticosus Hot plate, acetic acid induced writhing and tail flick methods were used to evaluate analgesic activity. The dose dependant increase in the analgesic effect was observed. RFF and RFL exhibited significant analgesic effect in hot plate test (Table 18, Graph 4); writhing test (Table 21, Graph 5) and tail flick test (Table 22, Graph 6). However the analgesic effect of RFR and RFS are comparatively less. Aspirin was used as standard drug. It is established that analgesic effect may follow the pathway of inhibit ing NFκB factor especially for the alcoholic leaves extracts of R. fruticosus (Kaur et al ., 2011). b) V. thapsus The analgesic effect was dose dependent. Leaves and fruit of V. thapsus showed significant analgesic effect as compared to root and stem using hot plate (Table 20, Graph 4) writhing (Table 21, Graph 5) and tail-flick method (Table 23, Graph 6). Literature revealed that Verbascoside an important constituent of V. thapsus may be considered for analgesic effect especially in acetic acid induced model (Nakamura et al ., 1997; Panchal et al ., 2010).

5. Diuretic activity a) R. fruticosus Diuretic activity was determined for a period of 3 hours. RFF at 100 and 300 mg exerted significant diuretic activity comparative to control group. Similarly mild diuretic activity

226 was observed at 300mg/kg for RFL. RFS showed significant results at 300 and 500 mg/kg. Hydrochlorothiazide was used as standard, showed diuretic index 8.5 (Table 24). b) V. thapsus Diuretic activity was determined following the same procedure as for R. fruticosus . We found that VTF at 100 and 300 mg/kg exerted significant diuretic action. There was no significant diuretic action for the rest of extracts applied. The details both for samples and standard are given in Table 24.

6. Gross Behavioural Studies a) R. fruticosus The gross behaviour findings of R. fruticosus fruit at the doses of 100, 300 and 500 mg/kg were almost normal. A slight increased in urination was observed at 100 and 300 mg/kg. The mice showed improved in their performance. The animals were more active perhaps it might be due the food nature of the drug that act as good source of ascorbic acid and other nutrients (Table 25-27).

In gross behavioural studies, the leaves extract of R. fruticosus at a dose of 100, 300 and 500 mg/kg the drug is safe and showed symptoms of alertness followed by relaxation or decreased activity (Table 28-30). Slight increased in urination was observed.

RFR at 100, 300 and 500 mg/kg were administered and the gross behavioural changes were observed (Table 31-33). The drug was safe but slight increase in light reflex was observed. The decreased in passivity was observed in other words motility was increased.

In these studies, extract of RFS at doses of 100, 300 and 500 mg/kg (Table 34-36) showed increased in motility with no toxic effects. As all four extracts RFF, RFL, RFR and RFS have almost the same activity with slight variation in their intensities. There were no irritations, aggression, vocalization, anaesthesia, tail lashing or exophthalmoses or enophthalmoses during the gross behavioural studies for R. fruticosus .

227 b) V. thapsus The gross behaviour effects of V. thapsus fruit at the doses of 100, 300 and 500 mg/kg were tabulated in Table 37-39. The fruit/pod extract exhibit slight decrease in pupil size, piloerection, passiveness, decrease in body done and decrease in pain response.

Similarly with the administration of leaves extracts (VTL) at the same doses 100, 300, 500 mg/kg abdominal discomfort was observed, the animals were calm, sitting to a side and motility was reduced (Table 40- 42).

The VTR at 100, 300 and 500 mg/kg showed decrease in pupil size, piloerection, decrease in alertness but not as prominent as for VTL (Table 43-45).

VTS had very mild effects slight irritation at higher doses and the rest of activities were almost normal it may be due to the fewer amount of active principles (Table 46-48).

7. Neuropharmacological Studies a) R. fruticosus After the administration of RFF there was increased in exploratory functions and slight anxiolytic effect was observed in open-field test and head dip test (Table 49, Graph 7) in comparison with control group.

In cage cross there was dose dependent stimulatory effect while in rearing the activity was lower at minimum dose but comparable to the normal at maximum dose that was 500 mg/kg so no sedative effect was observed (Table 51, Graph 8). There was no muscle relaxing effect observed for the fruit extract of R. fruticosus (Table 53). However strong antidepressant affect was observed comparable to diazepam (Table 55, Graph 9).

The exploratory motor activity increased as the dose of RFL increased in open-field test and there was anxiolytic effect in head dip test (Table 49, Graph 6) in comparison with control group. In cage cross and rearing the activity was slight calming at 100 and 300 mg/kg but normal at 500 mg/kg (Table 51, Graph 8). There was no muscle relaxing effect

228 during traction time test (Table 53). Antidepressant effect was observed in forced swimming test (Table 55, Graph 9).

RFR showed increased in exploratory in motor activity in open field test but motor activity decreases as the dose increases, however the results of head dip activity showed anxiolytic effect (Table 49, Graph 6).

There was slight calming effect in cage cross and rearing test with increase in dose (Table 51, Graph 8). The alertness was comparatively more so there was no decrease in the tone of muscle as illustrated by the findings in traction time test (Table 53). Antidepressant affect was observed in forced swimming test and the effect was almost comparable to Diazepam (Table 55, Graph 9).

In open field test the RFS treated mice showed almost normal response as compared to control at 100 mg/kg but as the dose increases the motor activity was reduced similarly increase in dose in head dip test produce anxiolytic effect (Table 49, Graph 6).

During cage cross test at 100 mg/kg the activity was normal but at dose 300 mg/kg slight calming effect was observed then at 500 mg/kg the mice become active and alert. In rearing test the exploratory effect increased with the dose (Table 51, Graph 8). Muscle relaxing effect was almost normal compared to RFF, RFL and RFR in traction test (Table 53). Slight antidepressant effect was observed (Table 55, Graph 9). b) V. thapsus During open field exploratory experiment the findings for VTF showed increased in motor performance however sedative effect was produced (Table 50, Graph 7) which was directly related to the dose.

In cage cross and rearing activity the calming effect was quit low as compared to Diazepam there was increased at 100 mg/kg then decreased at 300 mg/kg but below normal and again increased at 500 mg/kg (Table 52, Graph 8). The muscles were in nice

229 tone so there was decreased in traction time (Table 54). Highly significant antidepressant effect was there in forced swimming test (Table 56, Graph 9).

VTL treated animals showed decrease in the motor functions in open field test and sedative effect in head dip test in dose dependent passion (Table 50, Graph 7). In cage cross and rearing activity depressive effect decreased with the dose (Table 52, Graph 8). This depressive action did not effect muscles relaxation so dose dependent decrease in traction time was observed (Table 54). Highly significant antidepressant effect comparable to standard Diazepam was observed in forced swimming test (Table 56, Graph 9).

Dose dependent increased in motor exploratory behaviours was observed after administration of VTR in open field test however there was significant sedative effect at 100 and 300 mg/kg in head dip test but no sedative effect at 500 mg/kg (Table 50, Graph 7).

But there was depressive effect during cage cross activity and rearing test (Table 52, Graph 8). There was no muscle relaxing effect and the traction time was less than normal (Table 54). Slight antidepressant effect was observed in forced swimming test (Table 56, Graph 9).

In open field test the VTS treated mice showed rise in motor activity but the motor function decreased as the dose increased however in head dip test the sedative effect was quite less as observed for leaves and fruit of V. thapsus (Table 50, Graph 7).

Dose dependent decreased in stimulant effect was observed in cage cross test after treatment with VTS but still we found decrease in exploratory effect in rearing test (Table 52, Graph 8). There was no muscle relaxing effect observed in traction time test (Table 54). Slight antidepressant effect was observed (Table 56, Graph 9).

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8. Antimicrobial Studies i) Antibacterial activity a) R. fruticosus In our research study we found that RFF were quite effective against S. aureus, M. luteus, Citrobacter, B. subtilis and P. aeruginosa. The MIC values for these species were 20 µg (Table 57).

RFL were comparatively more active against E. coli and S. aureus with MIC of 20 µg against each strain. Growth of S. typhi , Citrobacter and P. aeruginosa were inhibited at larger doses and the MIC values were found to be 1000 µg (Table 58).

RFR showed activity against all tested bacteria in smaller doses except M. luteus and P. aeruginosa (Table 59).

The RFS were effective against all bacteria which were explained by its 20 µg MIC value against all species (Table 60). The zones of inhibition for standard drugs are also given in these tables. The Graph 10 showed the zone of inhibition for standards and samples. The antibacterial activity of RF various parts was compared with standard drugs.

Akiyama et al (2001) declared tannins for antibacterial action in plants in his studies (Doss et al ., 2009). Literature disclosed that flavonoids and tannins are among the important constituents of R. fruticosus (Gudej et al ., 2004). So the antibacterial action may be due to the presence of tannins (Djipa, et al ., 2000; Cavanagh et al ., 2003).

b) V. thapsus VTF showed no antibacterial action in low doses however at 1000 µg/disc it was quite effective against all bacteria (Table 61). S. aureus, P. mirabilis and B. subtilis were quite sensitive to low doses of VTL comparative to other tested species this was cleared from its MIC which is 20 µg against these bacteria (Table 62).

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B. subtilis growth was quite sensitive to low doses of VTR but E. coli was quite resistant and the rest of strains were inhibited at larger doses of 1000 and 2000 µg/disc (Table 63).

VTS showed inhibitory action against E. coli and Citrobacter at low doses and against S. typhi and M. luteus at larger doses but there was no inhibitory action against S. aureus, P. mirabilis, B. subtilis and P. aeruginosa (Table 64). The Graph 10 showed the zone of inhibition for standards and samples. The antibacterial activity of VTL were highest followed by VTF, which exhibited inhibitory action against all species but in larger doses. VTS were observed for least inhibitory action.

Slight antibacterial activity was reported by methanolic extract of leaves of V. thapsus (McCutcheon et al. , 1993; Turker et al ., 2001). Turker and Camper (2002) also reported slight antibacterial action by leaves extract against Klebsiella pneumoniae . But our studies for V. thapsus methanolic extracts showed significant antibacterial action comparative to previous antibacterial studies. The difference between the reported antibacterial action and our results may be attributed to the change in the geographical conditions and so metabolites. The antibacterial activity may be due to the presence of saponins in V. thapsus (Meurer-Grimes et al ., 1998; Panchal et al ., 2010). ii) Antifungal activity a) R. fruticosus Antifungal studies were carried out against S. cerevisiae, A. parasiticus, T. rubrum, M. phaseolinia, C. albican, F. solani, A. niger and A. effusus. Itraconazole and Amphoteracin B were used as standard drugs (Table 65). RFF, RFL, RFR and RFS exhibited no antifungal activity. b) V. thapsus Antifungal activity of VTF, VTL, VTR and VTS were explored against S. cerevisiae, A. parasiticus, T. rubrum, M. phaseolinia, C. albican, F. solani, A. niger and A. effusus. We observed no antifungal action of the tested extracts only zone of inhibition were observed for standard drugs (Table 65) .

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Literature revealed slight antifungal activity against human pathogenic fungi for methanolic extracts of leaves (McCutcheon et al. , 1994; Turker et al ., 2001) and also reduction in the growth of plant pathogenic fungi (Vogt et al ., 2010). Our results are totally different from the previous and this difference may be because of diversity in geography and hence constituents. The antifungal activity of some species of Scrophulariaceae was reported to be due to Ilwensisaponin A and C (Tatli et al ., 2003; Vogt et al ., 2010) and these compounds are yet not reported in V. thapsus or may be in negligible amount to be account for antifungal activity.

9. Toxicity studies Public opinion in general consider herbal medicine as safe because of natural origin however in real sense it is not true, various fatalities and illnesses were reported which required to be answered (Stewart et al .,1999; Veiga-Junior et al ., 2005; Escobara et al ., 2011). So both plants extracts were screened for their toxicities against worms, insects and brine shrimps.

9.1. Insecticidal activity a) R. fruticosus Insects and worms are sensitive to toxic substances. These techniques have some advantages for toxicity analysis. The simplicity, easily availability, low cost and can be carried out at normal bench space without sophisticated equipments.

Two insects, T. castaneum and S. oryzae were studied for repellent activity, paralysis and death at concentrations of 1, 5, 10, 25, 50, 75 and 100 mg for 24 hours. There was no significant toxicity for all four extracts of R. fruticosus (Table 66-69). Permithrin used as standard showed 100% mortality. b) V. thapsus Insects are very sensitive to certain natural agents so based on this approach we investigated the insecticidal activity of V. thapsus which is also an indication of toxicity or safety. T. castaneum and S. oryzae were used as models against different concentration

233 of V. thapsus. We found no significant insecticidal activity in other words we may say the drugs are safe at applied doses (Table 70-73). Permithrin used as standard showed 100% mortality at concentration of 235.9 μg/cm 2 (Table 66).

9.2. Antihelmintic activity a) R. fruticosus Antihelmintic activity was studied against Lumbricus terrestris . RFF applied at dose of 5 mg and 10 mg were safe however toxicity increased with the dose and death was observed within 6 to 12 hours (Table 74).

Paralytic effects were observed on worms treated with higher doses of RFL within 6 hours and death occurred within 24 hours. These studies suggest the lower doses safer (Table 75). Root extract resulted paralysis within 6 hours even at 25 mg/2ml however the animals were alive even after 24 hours. Death occurred at higher doses within six hours (Table 76).

RFS resulted in paralysis within 2 and death within 3 hours at 75 mg/2ml and 100 mg/2ml suggest it very toxic comparatively (Table 77). The standard drug Niclosimide was used in same doses as crude extract and resulted in paralysis within 35 minutes and death within 50 minutes even at lower doses (Table 82).

The toxicity symptoms of R. fruticosus were excitability with drug application, hyperactivity, jerks, sluggishness in movement, paralysis and finally death. But low doses were safe for all four extracts. b) V. thapsus Antihelmintic activity was studied against Lumbricus terrestris , VTF caused paralysis within ten minutes for higher doses and death within 1 hours however 5 mg and 10 mg caused paralysis in 30 minutes while death in the same duration as for higher doses (Table 78).

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VTL was more toxic to L. terrestris because it produced paralysis within 45 minutes and death in 75 minutes even at small doses (Table 79).

VTR was not as toxic as VTF or VTL because it produced paralysis within 10 hours and death in 24 hours for higher doses. There was no paralysis or death for 5, 10 and 25 mg (Table 80).

VTS was safe at lower (5 mg and 10 mg) doses however resulted in paralysis in 45 minutes and death in 60 minutes for 75 mg and 100 mg doses (Table 81). Keep in view the mean paralytic time and mean death time we reach at conclusion that VTL is most toxic followed by VTF then VTS and the least VTR.

9.3. Brine shrimp lethality test The brine shrimp assay provide information about general toxicity of the tested drug or extract and helpful in the prediction of anticancer agents (McLaughlin et al ., 1991; Turker and Camper, 2002). a) R. fruticosus Brine shrimp lethality test were carried out to evaluate the cytotoxic potential of various parts of R. fruticosus . The order of cytotoxic effect in our findings after LD 50 determination was RFR > RFL > RFS > RFF (Table 83-86, Graph 11).

Cyclophosphamide (LD 50 15.7 µg/ml) was used as standard (Table 91, Graph 11). As fruit is always consumed as food so it is very safe comparatively. However the lowest

LD 50 of RFR indicates the presence of some cytotoxic active constituent that may lead to a novel anticancer drug after bioactivity/safety studies. b) V. thapsus In Brine shrimp lethality test we found leaves and fruit extracts the toxic parts of V. thapsus in very low doses and VTR was the least toxic (Table 87-90, Graph 11).

Previously it is reported that methanolic extract of V. thapsus at 100, 300 and 500 mg/kg doses had no genotoxic or cytoxic effects (Escobara et al ., 2011). Brine shrimp and

235 radish seed bioassays proved that extracts of V. thapsus were toxic at higher (around 1000 µg/ml) doses (Turker and Camper, 2002). Our results corroborate the previous studies. The order of toxicity based on LD 50 are leaves > fruit/pod > VTS > VTR.

Positive correlation between brine shrimp toxicity and 9KB (human nasopharyngeal carcinoma) cytotoxicity existed (Meyer et al ., 1982). Our results in Brine shrimp toxicity test indicate clearly that we must proceed with advance anticancer studies especially fruit and leaves extracts of V. thapsus .

These toxicity findings provide preclinical knowledge which may help in therapeutic dose adjustment that would be safe and would be according to the potency of herbal drug.

Conclusion

Our studies on both plants R. fruticosus and V. thapsus that were collected from northern area of Pakistan, declared that leaves and fruit had significant antioxidant, anti- inflammatory, mild diuretic, antibacterial, anxiolytic, antidepressant and some potential of anthelmintic activity.

V. thapsus fruit and leaves were studied for the first time for anthelmintic activity and were found to have strong anthelmintic activity comparable to the standard. The brine shrimp lethality test confirmed leaves and fruit extracts the most cytotoxic. Their LD 50 values provide a clue to be studied further for cytotoxic constituent that may lead to the discovery of anticancer drug.

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