Asian Pac J Trop Biomed 2014; 4(1): 35-39 35

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Document heading doi:10.1016/S2221-1691(14)60205-7 2014 by the Asian Pacific Journal of Tropical Biomedicine. All rights reserved. 襃 Pharmacognostic studies of insect of O () livier

1 3 1,2 2 Savitri Shrestha , Vasuki Srinivas Kaushik , Ravi Shankara Birur Eshwarappa *, Sundara Rajan Subaramaihha , Latha 4 5 Muuaiah Ramanna , Dhananjaya Bhadrapura Lakkappa * 1Department of Chemistry, SBMJC, Jain University, Bangalore-560002, India 2Research Unit in Vrukshayurveda, A division of Centre for Advanced Studies in Biosciences, Jain University, Chamrajpete, Bangalore-560019, India 3National Referral Centre for Lead Projects in India (NRCLPI), Koramangala, Bangalore-560095, Karnataka, India 4Dayananda Sagar College of Applied Science, Kumarswamy layout, Bangalore, Karnataka, India 5Toxinology Lab, School of Chemical and Biotechnology (SCBT), SASTRA University, Thanjavur, Tamilnadu, India

PEER REVIEW ABSTRACT

Peer reviewer Objective: Quercus infectoria Q. To study the detailed pharmacognostic profile of of Olivier ( infectoria Dr. SB Benaka Prasad, Assistant Professor, olivier) (Fagaceae), an important medicinal used in the Indian system of medicine. Methods: Q. infectoria Department of Basic Science, School Samples of galls of were studied by macroscopical, microscopical, of Engineering and Technology, Jain physiochemical, phytochemical, fluorescence analysis and othjer methods for standardization as U K R niversity, anakapura, amanagar- recommended by WHO. 562112, India. Results: Macroscopically, the crude drug is globose with horny appearances on external surface Tel: 09986982138 (1 4 2 3 1 1 5 ) E-mail: [email protected] . - . cm in length and - . cm in diameter , with greyish-brown to brownish-black in colour externally and dark brown buff colored. Surface is smooth with numerous horny protuberances Comments giving rough touch, and with unpleasant odour. Microscopically, a wide zone of radially elongated This is an interesting study where the parenchyma cells between upper and lower epidermis were found. The vascular strands were present authors have undertaken to lay down at all places and radially elongated sclerides touched the lower epidermis. In physico-chemical the standards that could be useful for studies, the moisture, total ash, acid insoluble ash, alcohol soluble, water soluble, petroleum ether, establishing the authenticity of the chloroform extractive value and content were found to be 2.790, 5.020, 0.110, 38.780, 41.210, 0.402, drug material. 1.590 and 49.200 percentage respectively. Preliminary phytochemical screening showed the presence Details on Page 38 oConclusions:f phenols, flavonoids, steroids, triterpenes, , saponins and alkaloids. The results of the present study serve as a valuable source of information and provide suitable standards for identification of this medicinally important plant drug material for future investigations and applications. KEYWORDS Quercus infectoria , Gall extracts, Traditional medicine, Pharmacognostic study, Microscopic studies.

1. Introduction growing to 4 to 6 feet tall, crooked, with smooth and bright , acorn long and narrow, scaly and downy. The Quercus infectoria Q. infectoria ( ) (Family: Fagaceae) tree prefers partial shade or partial sun to full sun, and commonly known as gall oak, which is a small shrub found requires moist soil. It can grow in semi-shade or no shade in Greece, Asia Minor and Iran. It is a small tree or shrub and requires moist soil. The flowers are monoecious and *Corresponding author: Dr. BL Dhananjaya, Assistant Professor, School of chemical Article history: and Biotechnology (SCBT), SASTRA University, Thirumalaisamudram,Thanjavur-613401, Received 9 Oct 2013 Tamilnadu, India. Received in revised form 22 Oct, 2nd revised form 1 Nov, 3rd revised form 8 Nov 2013 Tel: 09597031796. Accepted 12 Dec 2013 E-mail: [email protected], [email protected] Available online 28 Jan 2014 Foundation Project: Supported by Tamil Nadu Science and Technology of Tamilnadu State Council for Science and Technology (Grant No. TNSCST/S&T Projects/VR/MS/2012- 13/203). Savitri Shrestha et al./Asian Pac J Trop Biomed 2014; 4(1): 35-39 36 2.2. Chemicals and instruments are pollinated by wind. The gall arising in the branches of ‘majuphal’ ‘machakai’ the tree is called as in Sanskrit and in Kannada (both are local languages in India). In India, Compound microscope, glass slides, cover slips, watch Q. infectoria the galls of are used since ages as a home glass and other common glass ware were the basic apparatus remedy for sore throat and chronic diarrhea in both rural and and instruments used for the study. Microphotographs urban areas. It is also used as an ingredient in Ayurvedic were taken using a Leica DMLS microscope attached with viz preparations[1]. Leitz MPS 32 camara. Solvents petroleum ether, benzene, Q. infectoria, n The gall, extracts of are extensively used in chloroform, acetone, ethanol (95%), -butanol and reagents viz traditional medication as karkatasringi for the preparation phloroglucinol, glycerine, HCl, chloral hydrate and sodium of Balachaturbhadra, Shringyadichurna, Karkatadichurna, hydroxide were procured from Sisco Research Laboratories Pistacia integerrima Kantkaryavaleha[2-4]. Traditionally, (SRL), Bangalore, India. is used as karkatasringi in the preparation of traditional Rhus succedanea Q. infectoria 2.3. Pharmacognostic study medications. Sometimes, and Pistacia integerrima are used as substitutes for in the preparation of karkatasringi, which is used in the treatment Fresh leaves and galls were taken for morphological and of coughs, phthisis, asthma, dysentery, skin diseases, histological studies. Coarse poweder was used to study the menorrhagia, intestinal hemorrhage, eczema, spermatorrhea microscopic characters, physicochemical and phytochemical intertrigo, impetigo, hemorrhages, chronic diarrhea and investigations. For microscopic studies, transverse sections trichomoniasis[5]. They are also used as an antidote, were prepared and stained as per standard procedure[20- febrifuge, ophthalmic, haemostatic agent, ointments and 22]. The powder microscopy was performed according to the Q. infectoria suppositories[6-8]. The galls of have been method of Khandelwal[22]. known to pocess medicinal properties, such as astringent, 2.4. Physicochemcial and phytochemical analysis anti-inflammatory, antiviral, antidiabetic, larvicidal, antibacterial, antiulcerogenic and gastroprotective activities[9-16]. The gastroprotective effect against Physicochemical values such as % of ash values and ethanol-induced gastric damage is also attributed to the extractive values were determined according to the well Quercus ilex aqueous extract from [17]. Ethanolic established protocols[23,24]. Phytochemical screening Quercus aegilops extracts of and was carried out using standard procedure described by have been showed have a very high curative rate after Khandelwel[22]. ethanol-induced gastric damage in rats suggesting 2.5. Florescence analysis their use in treatment of stomach pain[18]. In view of its diverse medicinal applications and in order to ensure the quality, authenticity and assay[19], and in view of Powdered gall extracts were treated with various chemical lack of pharmacognostic study, the present investigation reagents and exposed to visible, ultraviolet light (short UV) to Q. was undertaken with an objective to evaluate galls of study their fluoresce behaviour[25,26]. infectoria on various pharmacognostic parameters, such as macroscopic, microscopic, physiochemical, fluorescence 3. Results and phytochemical studies of the plant. 3.1. Macroscopic characteristics 2. Materials and methods Q. infectoria is a small tree or shrub growing to 4 to 6 feet 2.1. Plant material tall, crooked, with smooth and bright leaves, acorn long and narrow, scaly and downy. The branches often slender Q. infectoria The galls of were collected locally from and drooping. Leaves are elliptical, glabrescent and up Bangalore, Karnataka (Southern India) in May 2010 and to 4 cm long. Petioles are up to 4 mm long. Flowers are in authenticated at Regional Research Institute (Ayurveda), axillary fascicles, pedicels filiform. are baccate and

Pune. And a voucher specimen of the plant was deposited 8 mm in diameter. It becomes black while ripening. Root is for future reference. The collected sample was dried cylindrical, branched and shows fibrous fracture, 6-10 cm under shade, packed in a paper bag and stored at ambient long and 4-8 mm in thickness. The crude drug is globose temperature until use. with horny appearances on external surface with size of Savitri Shrestha et al./Asian Pac J Trop Biomed 2014; 4(1): 35-39 37

1.4-2.3 cm in length and 1-1.5 cm in diameter, with Greyish- B C brown to brownish-black in colour externally and dark A brown buff coloured. Surface is smooth with numerous horny protuberances giving rough touch, and with unpleasant odour (Table 1). The galls are globular (2 inch), with uneven surface with pores (indicates infection), with hallow structures and Tableinner s1urface is yellow (Figure 1). MacroscopQ.ic infectoriacharacter istics of crude drug and its powder form of gall extract of Olivier. Q. infectoria Olivier Characteristics Crude Drug Shape Globose with horny appearances on external surface Size 1.4-2.3 cm in length and 1-1.5 cm in diameter Surface Smooth with numerous horny protuberances giving rough touch D E F Colour Greyish-brown to brownish-black in colour externally and dark brown buff coloured Odour Not characteristic Taste Bitter astringent but at end sweetish sensation Fracture Short granular Powder Nature Mixture of coarse and fine Colour Creamish-white Touch Rough to smooth Odour Not characteristic Taste Bitter astringent

Figure 2. Q. infectoria. Microscopic characteristics of gall of A B A: Upper epidermis, B: Lower epidermis, C: Outline of lowermost portion, D: Parenchyma cells in middle portion, E: Upper epidermis, hypodermis and parenchyma cells, F: Radially elongated sclereids with septate wall. 3.3. Powder characters

Mixture of coarse and fine, with creamish-white colour. It is C D with no characteristic odour with bitter taste (Table 1). 3.4. Preliminary phytochemical screening

Preliminary phytochemical screening revealed the presence of saponins, alkaloids, tannins, glycosides, triterpenes, sterols, Figure 1. Q. infectoria phenolic compounds, carbohydrates, and flavonoids in various Macroscopic characteristics of gall extract of . extracts (Table 2) A: Crude drug; B: Powder form, C: External surface showing pore of insect Table 2 exit, D: Internal surface in broken drugs. Q. infectoria. Preliminary phytochemical screening of leaf gall of 3.2. Microscopic characteristics Chemical Water Alcohol Petroleum Chloroform constituents extract extract ether extract extract Phenols + + - - Usually upper and lower epidermis cover the whole Flavonoids + + - - section, but with the upper epidermis ruptures is replaced Steroids - - + + 1 2 by a metaderm composed of - layers of suberised cells Triterpenes - + + + (Figure 2A). This is followed by a wide zone of parenchyma Tannins + + - - (Figure 2D), the cells of middle and inner layers being Saponins + + - - larger in size and somewhat radially elongated (Figure 2F). Alkaloids + + - + Number of these cells contain tannin and many of them are Glycosides - - - - Carbohydrates + + - - having pitted wall. However, morphology of gall shows the + Denotes the presence of the respective class of compounds. pores which means exist of insects, but at places remains of insects, dark brown in colour and few remains of larva 3.5. Physiochemical constants are also observed (Figure 1C and 1D). Vascular strands are present at places. Radially elongated sclereids were found The percentage of total ash, acid insoluble ash, sulphated touching the lower epidermis (Figure 2F). They are 4-7 ash and water soluble ash were shown in Table 3. The ash layered and are interrupted with parenchyma cells at places. values of a drug give an idea of the earthy matter or the Deposition of colouring matter was more concentrated inorganic composition and other impurities present along with towards the lower epidermis (Figure 2C and 2E). the drug. The loss on drying and foreign matter was 9.50 and Savitri Shrestha et al./Asian Pac J Trop Biomed 2014; 4(1): 35-39 38 infectoria 0.10, respectively. The extractive values are primarily useful will provide useful information for its identification for the determination of exhausted or adulterated drugs. The of this medicinally important plant. Macro, micro and water soluble, alcohol soluble and ether soluble extractive physiochemical standards discussed here can be considered values were shown in Table 4 and the results of fluorescence as the identified parameters to substantiate and authenticate analysis of the drug powder were presented in Table 5. the drug. Thus exploring the usefulness of pharmacognostic Table 3 evaluation to substantiate and authenticate drug. Q. infectoria. Hysiochemical parameters of leaf gall of Parameters Value Conflict of interest statement Foreign matter (% w/w) 0.1 Loss on drying (% w/w) 9.5 Total ash value (% w/w) 5.02 We declare that we have no conflict of interest. Acid insoluble ash (% w/w) 0.11 Water soluble ash (% w/w) 2.22 Acknowledgements Sulphated ash (% w/w) 0.21 Swelling index (mL) 2 Foaming index (mL) - We acknowledge Dr. R. Chenraj Jain, President, Jain Table 4 University Trust., Dr. N Sundararajan, Vice Chancellor, Q. infectoria Jain University and Prof. K.S. Shantamani, Chief Mentor, Extractive values of leaf gall of . Jain University, Bangalore for their kind support and Extract Yield (% w/w) encouragement. DBL thank SASTRA University and Jain Water 41.215%, 15.47% University, for providing facilities for carrying out this work. Methaholic 38.785%, 21.29% The Financial supported by SASTRA University to BLD in the Chloroform 1.59% form of TRR research grants is gratefully acknowledged. DBL Pet ether 0.40% Table 5 acknowledges the financial support by Tamil Nadu Science Q. infectoria T (G N TNSCST S T P VR MS 2012 Florescence analysis of powdered gall extract of . and echnology rant o. / & rojects/ / / - Treatment Visible Long Wavelength UV Light Short Wavelength UV Light 13/203) of Tamilnadu State Council for Science and Technology 1N NaOH (Aqueous) Royal Red Brown Dark Brown to carry out this work. 1N NaOH (Alcoholic) Light Golden Yellow Fluorescent Yellow Fluorescent Dull Green 1N HCl Orange Yellow Light Brown Greenish Yellow Comments Acetic Acid Yellow Fluorescent Light Green Fluorescent Light Green Ethyl Alcohol Yellow Fluorescent Orange Fluorescent Dull Green H2SO4 (50%) Golden Yellow Light Brown Fluorescent Green Background Powder as such Reddish-brown Yellow Orange Yellow Many plant drugs used in medicinal folklore are being 4. Discussion evaluate, due to their specific healing properties, health action and non-toxic effects. In this regard, as galls of Q. infectoria Olivera are currently being used in the treatment of various For measuring the quality, purity and ensuring the disease conditions, the pharmacognostic parameters, such as authenticity of the drugs used, standardization is very macroscopic, microscopic, physiochemical, fluorescence and essential. The cost effective and time saving method is the phytochemical studies of the drug are the most cost effective microscopic evaluation to establish the right identification and time saving methods to identify parameters to substantiate of the drug material. As Karkatashringiis are currently being and authenticate the drug. used in the treatment of various disease conditions, and till Research frontiers date therQ.e iinfectorias no report on standardization of the material (Galls of ). The present work was undertaken to Studies are being performed in order to establish the lay down the standards that could be useful for establishing authenticity of standard drug that is used to treat various the authenticity of the drug material. The micro and macro diseases. The total ash value, fluresecence analysis and standards obtained here can be identifying parameters to extractive values will be helpful in identification and substantiate and authenticate the drug. The preliminary authentication of the plant material. phytochemical screening will be useful in finding the Related reports chemical nature of the drug. In this study, the preliminary phytochemical screening ascertained the presence of steroids, The powder microscopy was performed according to the triterpenes, tannins, saponins and alkaloids. The total ash method of Khandelwal[22]. Physicochemical values such as % value, fluoresecence analysis and extractive values will of ash values and extractive values were determined according be helpful in identification and authentication of the plant to the well established protocols[23,24]. Phytochemical material[27,28] along with the microscopic method, which is the screening was carried out using standard procedure described cheapest method to establish the correct identification of the by Khandelwel[22]. Powdered gall extracts were treated with source material[29]. Also the extractive methods are useful to various chemical reagents and exposed to visible, ultraviolet [30] T ( UV) [25,26] evaluate the chemical constituents of the crude drug . hQ.e light short to study their fluoresce behaviour . present study on pharmacognostical evaluation of leaf gall of Savitri Shrestha et al./Asian Pac J Trop Biomed 2014; 4(1): 35-39 39 Innovations and breakthroughs Quercus inhibitory activity of hexagalloyl glucose from the galls of infectoria Planta Med 66 Pharmacogostic studies regarding galls are very scarce. This . 2000; : 273-274. [12] R edwane A, Lazrek HB, Bouallam S, Markouk M, Amarouch H, study has established the micro and macro standards obtained Quercus lusitanica T Jana M. Larvicidal activity of extracts from var. for the first time, preliminary phytochemical screening. he J Ethnopharmacol 79 total ash value, fluresecence analysis and extractive values infectoria galls (Oliv.). 2002; : 261-263. [13] B asri DF, Fan SH. 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