Indian Journal of Natural Products and Resources Vol. 4(1), March 2013, pp. 88-95

Pharmacognostical evaluation of root of Gumma ( Spreng.)

Mohammad Yusuf Ansari1, Abdul Wadud1*, Ehteshamudddin1 and Hamida Bano2 1Department of Ilmul Advia, National Institute of Unani Medicine, Kottigepalya, Magadi Main Road, Bangalore-560 091, Karnataka, 2HAH Unani Medical College, Idgah Road, Dewas, Madhya Pradesh, India

Received 20 June 2011; Accepted 6 August 2012

Gumma (Leucas cephalotes Spreng.) belonging to family , primarily a folk drug, is also used in Ayurveda and Unani medicine in India and in adjacent countries for its varied therapeutic properties such as stimulant, diaphoretic, antiseptic, laxative, anthelminthic, insecticidal, germicidal, fungicidal, emmenogogue, expectorant and antipyretic. Its root is particularly useful in regularization of menstrual cycle, tuberculosis and dysentery. In view of insufficient data necessary to set up proper identification of the whole and its roots and to provide referential information for checking adulteration and substitution, root of this plant has been evaluated on pharmacognostical parameters by means of anatomical, physico-chemical and preliminary phytochemical studies, HPLC and UV-Vis spectrophotometry. Keywords: Gumma, Leucas cephalotes, Anatomical studies, Physico-chemical studies, HPLC, Spectrophotometery. IPC code;Int. cl. (2011.01)—A61K 36/00

Introduction some physical and chemical operations on the drug In spite of the reality that herbal drugs can be sample prior to the actual analysis. A blend of safely used only if their standards i.e. safety, efficacy classical methods and latest analytical techniques are and quality are up to the mark, these important values advantageous in the study of herbal drugs. have not been looked up appropriately. In view of the Gumma (Leucas cephalotes Spreng.) belonging growing interest in herbal drugs that has called for to the family Lamiaceae is an annual herb and greater exactitude in appraisal, it becomes crucial to an upland rainy season weed commonly found ascertain standard samples of single herbal drugs for in roadsides, meadows, waste lands and cultivated future reference1. The rapid development in different grounds throughout the greater part of India4-12 aspects concerning to crude herbal drugs, in recent (Plate 1). The plant as a whole and its various years, has necessitated a systematic approach to parts alone are used medicinally. It possesses stimulant, evaluate these drugs in modern pharmacognosy with a laxative13, diaphoretic14, antiseptic15, antihelmenthic16, methodical approach to qualitative and quantitative insecticidal17,18, germicidal19, fungicidal20, emmenogogue21, evaluation by means of appropriate methods as per expectorant and antipyretic22 properties. Powdered WHO guidelines2,3. root is used as one of the ingredient of compound Analytical instruments play important role in the remedy to treat tuberculosis. To treat dysentery it is production and evaluation of new products in the given with edible oil once daily23. Root of Gumma safety of consumers and the environment. The use of with Chota Kulpha [Trichodesma indicum (L.) R. Br.] sophisticated instruments, in present days, is a fascinating part of chemical analysis. Though, it is necessary to use several instrumental techniques to obtain the information required to solve analytical problems still worth of classical methods can’t be underestimated. Therefore, it is necessary to perform

—————— *Correspondent author : E-mail: [email protected] Phone: 080-23584260 Fax: 080-23580725 Plate 1—a. Whole plant, b. Root ‘ ANSARI et al: PHARMACOGNOSTICAL EVALUATION OF ROOT OF GUMMA 89

and Kali Musli (Curculigo orchioides Gaertn.) is used Microscopic studies for the treatment of menorrhagia. Use of powder of Transverse section (T.S.) of the root was prepared 29 root with rice water regularizes menses23. Some recent according to the method described by Johnson . ethno pharmacological studies have shown the plant Small piece of the material was kept in a vial and to be hypoglycaemic, hypolipidaemic, antioxidant24, fixed in 50 % ethanol (v/v). After two days, washed anti-inflammatory25, antiprotozoal26 and hepatoprotective27. with the same fluid and dehydrated with ascending The whole plant reported to contain new labdane-, series of ethanol (50, 70, 95 and 100%), dipped in norlabdane- and abietane-type diterpenes named each series for about 2 h. Thereafter, passed through leucasdins A (1), B (2) and C (3), respectively, and the descending series of absolute alcohol and xylol two protostane-type triterpenes named leucastrins (75:25, 50:50; 25:75) and finally washed with xylol. A (4) and B (5) were isolated, together with a In xylol the infiltration was carried out by gradual known triterpene, oleanolic acid, five sterols, addition of paraffin wax at room temperature till the 7-oxositosterol, 7-oxostigmasterol, 7α-hydroxysitosterol, xylol was saturated. The vials were kept in an oven at o o 7α-hydroxystigmasterol and stigmasterol, and eight 35 C + 2 C, the contents were occasionally stirred and flavones, 5-hydroxy-7,4'-dimethoxyflavone, pillion, wax was added till the solution saturated. After 24 h, o o gonzalitosin I, tricin, cosmosin, apigenin 7-O-β-D- the temperature of the oven was raised to 60 C + 2 C, (6-O-p-coumaroyl) glucopyranoside, anisofolin A and and the addition of wax was continued till the smell of luteolin 4'-O-β-D-glucuronopyranoside28. xylol vanished. The mixture of xylol and wax was Though this plant possesses a number of poured off after three hours and pure melted wax was therapeutic properties, very little description is added. The vials were left inside the oven for 24 h and available in classical Unani literature and that too the material was tested for complete infiltration by compiled in Indian subcontinent. Also, it has not been cutting with a safety razor. The contents were poured studied scientifically to a great extent to support the into a dish containing melted wax and materials identification of genuine sample. Keeping these points arranged in rows. The dish was dipped into beaker in view present work was undertaken to study containing cold water. The rectangular blocks were pharmacognosy of root of Gumma. made after trimming suitable size. Soft wax applied on the both the surfaces and then fixed in block Materials and Methods holder. For sections the block was fixed in the block Collection and authentication of the plant holder which was fixed in clamp of microtome. The The fresh plant was collected from the forest of microscale of microtome was set at 10 micron. The Satpura range of Burhanpur (M P), India in the month wheel was moved with steady even stokes. The first of July. The plant was identified and authenticated by few sections were discarded until a ribbon was made. botanists of National Ayurveda Dietetic Research The slide was placed on the table and flooded Institute, Bangalore vide authentication certificate no. immediately with 03% formalin in distilled water. Drug Authentication/SMPU/NADRI/ BNG/2009-10/896. The ribbon was then kept on the slide with the help of Fresh material was used for anatomical studies a wet scalpel, and the slide was put on a warm plate. whereas shade dried material was powdered in The temperature was not allowed to exceed 43°C. electric grinder for physico-chemical, phytochemical, After few minutes the slide was removed and kept to HPLC and spectrophotometric studies. cool up to room temperature. The water was drained Preparation of extracts off by tilting the slide. After drying the slide, it was The study was carried out in the pharmacognosy placed in xylol for 5 minutes. The slides were then laboratory and Central Instrumentation Facility withdrawn slowly and transferred to a coupling jar, laboratory (CIF) of National Institute of Unani containing equal volumes of absolute alcohol and Medicine (NIUM). Coarse powder of air dried root xylol for five minutes then into 75:25 ethanol and (100 g) was subjected to Soxhlet apparatus for 8 h for xylol solution and finally in absolute alcohol solution continuous hot extraction with distilled water, later transferred to 95, 70, and 50% ethanol methanol, acetone, diethyl ether, petroleum ether and successively in each for five minutes. The section chloroform, separately. The extracts were filtered and were then kept in saffranin (in 50% ethanol) for five the filtrate was evaporated to dryness. The percentage minutes and transferred to 50, 70, 95 and 100% yield of each solvent was calculated with reference to ethanol in ascending order keeping each for the air dried drug. 2-3 minutes. The slides were again transferred to 90 INDIAN J NAT PROD RESOUR, MARCH 2013

clove oil and ethanol mixture (75:25) then in fast 0.5 mL/minutes using methanol: water (70:30) as green prepared in clove oil and xylol mixture (75:25) mobile phase solvent, under a pressure of 100 f/sq cm, for 2 minutes in each. It was washed through run time of 10 minute and an injection volume of xylol and finally mounted in DPX mountant. 20 µL. at 240, 205, 254, and 238 nm. Analyst The section observed under microscope with 1.4 software was used to control all the parameters. different magnifications i.e. 50, 100 and 450. Photomicrographs were taken by using digital camera Spectrophotometry (Sony, Japan) attached to microscope. Various cells Spectrum scan curves of aqueous and methanol were measured (micrometry) with the help of extracts of root were obtained by using UV-Vis micrometer (stage micrometer and ocular micrometer) Spectrophotometer 3000 (Labindia) at room under 100 magnifications by the method of Trease temperature according to the method given in the and Evans. Isolated elements were also studied by instruction manual. After pre-heat time, UV-Vis reported method of Trease and Evans 30. spectrophotometer was assessed to spectrum scanning mode. The parameters were set, the photometric Physico-chemical studies mode was assessed to Abs, scanning speed was For physico-chemical studies, ash and extractive set as middle and the wave length range was set to values and pH were measured by the method 190-660 nm. Baseline correction was performed described in British pharmacopoeia31. Moisture with the blank cell and then samples of extracts of content was determined by the method of Jenkin drug were scanned. et al32. Florescence analysis of the powdered drug was carried out according to the method of Kokoshi Results et al33. On a micro slide, one mg powdered drug was T.S of root showed almost circular structure placed and exposed to UV and day light to observe composed of thick walled, elongated, multilayered fluorescent characteristics of the drug. Thereafter epidermal cells and epithelial cells. The secondary same amount of drug was placed on different micro cortex was composed of tangentially arranged, slides and treated separately with 1N HCl, 1N NaOH, elongated, thin walled, irregular, multilayered 1N NaOH in one mL of methanol, 50% KOH, 50% parenchymatous cells. Beneath the cortex, single H2SO4, concentrated H2SO4, 50% HNO3, concentrated layered non-lignified pericycle was observed. Phloem HNO3, acetic acid, iodine solution distilled water, consisted of sieve tissue embedded in chloroform, acetone and picric acid, each one mL. parenchymatous cells. Xylem region consisted of The slides were exposed to UV (short wave length) xylem vessels, xylem parenchyma and medullary and day light to observe fluorescent characteristics rays. Xylem vessels were found scattered, lignified, of the powder. reticulate, annular, isolated. Xylem parenchyma closely resembled to the phloem. The medullary rays Preliminary phytochemical studies made up of parenchymatuos cells ran radially and Preliminary phytochemical screening for extended from the pith to cortex, uniseriate to qualitative detection of various phytochemicals was biseriate and very similar in character to the done by using the method of Bhattacharji and Das34. surrounding parenchyma (Plate 2 a-d). Micrometry of the cells is given in Table 1. Study of isolated High Performance Liquid Chromatography (HPLC) elements showed presence of sclerenchymatous HPLC of aqueous extract of root was run on fibers, vascular bundles, tracheids, reticulate vessel, an ultra fast liquid chromatography (UFLC) system oil globules, crystal, and scalariform vessel (Plate 3 a-h). with a LC-20AD pump and 20A auto-sampler (Shimadzu, Japan). Phenomenex Luna C18 (2) column Physico-chemical studies (250 X 4.6 mm id) 5 micron was maintained at The physicochemical parameters such as total ash, 40oC. Mobile phase solvents were filtered through acid insoluble ash, water soluble ash and 0.45 µ membrane Millipore, PVDF under vacuum. water insoluble ash were estimated as 7.37 ± 0.26, The sample for analysis was filtered through 0.22 µ 2.89 ± 0.18, 1.22 ± 0.01 and 6.24 ± 0.31, respectively. membrane. The mobile phase A, solvent was double Extractive values were estimated as 3.53 ± 0.03, 2.37 distilled water. The mobile phase B, solvent was ± 0.02, 3.28 ± 0.01, 6.48 ± 0.12 10.17 ± 0.26 and HPLC analytical grade methanol. The flow rate was 15.22 ± 0.48 in petroleum ether, diethyl ether, ANSARI et al: PHARMACOGNOSTICAL EVALUATION OF ROOT OF GUMMA 91

Plate 2—T.S. of root at various magnifications (a. 50%, b. 100%, c. & d. 450 %)

Table 1—Micrometry of root of Leucas cephalotes Spreng. by treating with various chemical reagents and observed in day and ultraviolet light (Table 2). S. No. Cells Measurement (µm) (Range)

1. Epidermal cells 23.06 – 27.67 – 34.59 (L) Phytochemical studies 2. Epithelial cells <11.53 (L) The phytoconstituents were screened in the 3. Parenchymatous 23.06 – 39.20 – 57.65 (T), extracts taken in water, methanol, acetone, diethyl cells <11.53 (L X B) ether, petroleum ether and chloroform extracts. 4. Pericycle 11.53 (T) The screening exhibited presence of glycoside, 5. Xylem vessels 11.53 –20.75 – 34.59 (L) carbohydrate, phytosterols, tannins, protein and amino 6. Medulary rays 403.55 – 587.63 – 807.1 (L) acids and fixed oil (Table 3). 7. Cells surrounding 8.12 – 12.47 – 23.06 × 8.12 – medulary rays 10.16 – 11.53 (L X B) High performance Liquid Chromatography (HPLC) L=Length, B=Breadth, T=Thickness HPLC of aqueous extract of root (Aq. ext LC-R) showed four peaks in chromatogram. From refractory chloroform, acetone, methanol and distilled water, time of 1.48 min first peak was observed at 1.483 min respectively. Moisture content was found to be 5 ± with peak area, peak height of 10.961 mV and 0.31 and pH in 1 and 10% solution was found to 7.21 1.1.358. Total of four peaks were observed. The wave ± 0.05 and 6.48 ± 0.15 (n=5). Result of fluorescence range selected for scanning was the entire UV and analysis of the powder of root carried out separately visible range. The results and the parameters applied 92 INDIAN J NAT PROD RESOUR, MARCH 2013

Plate 3—Isolated elements of root at various percentage of magnifications (50, 100 & 450) : (a) Reticulated fibres (450), (b) A- Reticulated vessel, B-Crystal, C- Fibre (50), (c) A-Crystal, B-Oil globule (50), (d) A- Crystals, B- Scalariform vessels (50), (e) Oil globule (100), (f) Compound vessels (450), (g) Compound vessel (100) , (h) Trachieds (450).

Table 2—Fluorescence analysis of powder Table 3—Qualitative phytochemical analysis

S. No Tests Observations S. No. Constituents Extracts Under day light Under U/V light Aqueo Ace Pet. Ether Metha Chloro 1. Powder Light golden rod Dark golden rod us tone ether nol form 2. Powder+1NHCL Creamy yellowish Yellowish green white 1. Alkaloids ------3. Powder+1NNaOH Brilliant green Lime 2. Glycosides + ------+ -- 4. Powder+1NNaOH+ Dark golden rod Yellowish green 3. Carbo- + ------+ -- Methanol hydrates 5. Powder+50%KOH Golden rod Medium spring 4. Steroids -- + + + + --

6. Powder+50%H2SO4 Golden rod Medium spring 5. Phenolic + ------+ -- green compounds

7. Powder+Conc. H2SO4 Dark red Dark slate g 6. Tannins + ------+ -- 8. Powder+50% HNO3 Orange Lime 7. Fixed oil -- -- + + -- -- 9 Powder+ Conc. HNO3 Brilliant orange Yellowish 8. Saponin ------10. Powder+ Acetic Acid Saddle brown Spring 9. Amino acids + ------+ -- 11. Powder+ Iodine solution Burgundy Yellowish green 10. Flavonoids ------12. Powder+ Distilled water Sandy brown Medium spring + =Present; -- =Absent 13. Powder+ Chloroform Golden rod Yellowish green 14. Powder+ Acetone Golden rod Lime efficacy and stability of herbal preparations, is 15. Powder+ Picric Acid Orange Yellowish green frequently problematic but these problems can be are summarized in Fig.1. The solvent systems are solved by making suitable strategies for the described in the methods section. standardization of crude drugs and their preparations in order to maintain the homogeneity of the plant Spectrophotometery analysis material as much as possible. The microscopical The aqueous and methonolic extracts of root characters of a plant material such as types and (Aq ext LC-R) gave five peaks and two valleys arrangements of different cells, the typical shape of whereas methanolic extract of root (Met Ext LC-R) trichomes, vascular bundle and other cells and gave three peaks and three valleys in spectrum micrometry are not only helpful but also essential for scanning (Fig. 2). the identification of the material specially for those materials which are avilable in pieces. Therefore, Discussion often used in association with other analytical Though, the quality of raw materials which plays methods35. Physicochemical standards usually include key role in the confirmation of the purity, safety, ash values, extractive values, moisture content, pH ANSARI et al: PHARMACOGNOSTICAL EVALUATION OF ROOT OF GUMMA 93

and fluorescence analysis of powdered drug. Qualitative analysis of chemical constituents is generally widely accepted parameter. Extractive value in different solvents is a valuable test to check the quality of drug. It is the amount of extract that a drug yields to a certain constituents or a group of related constituents that a drug contains. The specific solvent is used to extract the specific phytochemical. The amount of extract in a particular substance plays important role in establishing the index of the purity. Any adulteration or substitution may cause change in extractive value35. Another important parameter is the ash value which helps in detection of adulteration and impurities of a drug. It can also detect the nature of added material in herbal drug36. An excess of water in medicinal plant material encourages microbial growth and deterioration following hydrolysis. Estimation of moisture content is important for the material which absorbs moisture easily or deteriorates quickly in the presence of water and therefore, quality and efficacy of the drug is compromised. Thus, estimation of moisture content may be a good parameter for checking the purity of the drug36. This parameter was also applied in the study. Herbal drugs are generally used in powder form which makes them more susceptible for adulteration. This problem can be solved by observing the powder of the drug under day light and U/V light after treating the powder with different chemicals because the fluorescence characters of different drugs

are diagnostic. It helps in detecting the adulteration. Figure. 1—HPLC of aqueous extract This parameter was also applied in the study. Result is

Figure 2—Spectrum scanning curves of aqueous and methanol extract 94 INDIAN J NAT PROD RESOUR, MARCH 2013

shown in Table 1. Phytochemical which include 3 Kokate CK, Practical Pharmacognosy (Vallabh Prakashan, mainly alkaloids, glycosides, essential oil, tannins, Delhi), 2007, 1. 4 Khanam M, Abul Hasan MD, A critical study of the genus resins, flavonoids, etc. are receptive parameters for Leucas R. Br. (Lamiaceae) from Bangladesh, Bangladesh J standardization. These phytochemical not only vary Plant Taxon, 2005, 12 (1), 1-10. from to species but also differ in different 5 Saxena NB and Saxena S, Plant , 5th Edn, Pragati samples of the same drug therefore, it can be used as a Prakashan, Meerut, 2006, 409-410. sensitive parameter in the quality control of drugs,. 6 Rajan SS, College Botany, Vol. III, Himalaya Publishing House, Mumbai, 2004, 138-145, 544. Our study demonstrated presence of glycoside, 7 Oudhia P, Dronapusphi or Gumma (Leucas cephaloes): carbohydrate, phytosterols, tannins, protein and amino A useful weed, Raipur, www.celestine-india.com. (cited on acids and fixed oil (Table 2). April 29, 2009). Recently, it has been possible to use sophisticated 8 Pullaiah T, Ramakrishnaiah, Sandhya RS and Rao PN, Flora of Guntur District, Andhra Pradesh, Regency publication analytical methods such as HPLC, HPTLC, UV/VIS- New Delhi, 2000, 287. spectrophotometry, etc. for isolation and identification 9 Khare CP, Indian Medicinal Plant: An Illustrated Dictionary of chemical constituents present in the drug with high (Springer Science + Business media, New York), 2007, 372. end results. HPLC is a fast, sensitive and most 10 Pullaiah T, Biodiversity in India, Vol. VI, Regency preferred chromatographic technique for routine assay publication, New Delhi, 2006, 351. 11 Chauhan NS, Medicinal and Aromatic of Himachal of new drug as well as determination of adulterant of Pradesh, Indus Publishing Company, New Delhi, 1999, 260. established drugs. UV/VIS absorption technique may 12 Pullaiah T and Silar MM, Flora of Ranga Reddi District, be used for analysis of a variety of natural products. Andhra Pradesh, India, Regency publication, New Delhi, In the present study HPLC of aqueous extract and 2000, 175. 13 Kirtikar KR and Basu BD, Indian Medicinal Plants with UV/VIS-spectrophotometry of aqueous and methanol nd Illustrations, Vol. IX, 2 Edn, Oriental Enterprises, Dehra extracts of the root have been carried out. Since Dun, 2003, 2779-2781. marker compounds for the plant have not yet been 14 Anonymous, The Useful Plants of India, National Institute of established, the present study serves as a lead in Science Communication and Information Resources, New HPLC studies of Gumma. It may serve as preliminary Delhi, 2000, 326. 15 Anonymous, The Wealth of India- A Dictionary of Indian study and may help in the further refined studies. Raw Materials, National Institute of Science Communication Detailed results are given in Figs. 1 and 2. No and Information Resources, Council of Scientific and convincing data for the root of Gumma (L. cephalotes Industrial Research, New, Delhi, 2003, Vol. IV, 79-80. Spreng.) on the above parameters are available to 16 Pullaiah T, Encyclopedia of World Medicinal Plants, Vol. I, compare our findings therefore, our finding may be Regency publication, New Delhi, 2006, 1239. 17 Dymock W, Warden CJH and Hooper D, Pharmacographia considered as an addition to the existing knowledge. Indica: A History of the Principal Drugs of Vegetable Origin,

Vol. III, Srishti Book Distributors, New Delhi, 2005, 123- Conclusion 124. In the light of the present study it can be concluded 18 Chopra RN, Glossary of Indian Medicinal Plants, National that the findings can serve as source of information to Institute of Science Communication and Information ascertain the authenticity and standardization of the Resources, New Delhi, 2002, 153. available sample of the drug. 19 Anonymous, Hamdard Pharmacopoeia of Eastern Medicine, Sri Satguru Publication, Delhi, 1997, 7, 42, 50. Acknowledgments 20 Chatterjee A and Prakashi SC, The Treatise on Indian The authors are thankful to the department of Medicinal Plants, Vol. V, National Institute of Science Communication and Information Resources, New Delhi), AYUSH, M/O Health & F/W, Govt. of India for 2003, 17-18. providing financial assistance and to Prof. M. A. Jafri, 21 Rastogi RP and Mehrotra BN, Compendium of Indian Director, National institute of Unani Medicine for Medicinal Plants Vol. II, Central Drug Research Institute, providing facilities for research work. Lucknow, 1999, 414. 22 Anonymous, Medicinal Plants in Folklores of Bihar and References Orissa, CCRUM, Ministry of Health & Family Welfare, 1 Patra KC, Pareta SK, Harwansh R and Kumar KJ, Govt. of India, New Delhi, 2001, 314. Traditional approach towards standardization of herbal 23 Bavarva JH and Narasimhacharya AV, Leucas cephalotes medicine- A Review, J Pharmaceut Sci Tech , 2010, 2 (11), regulates carbohydrate and lipid metabolism and improves 372-379. antioxidant status in IDDM and NIDDM rats, 2 Shinde V and Dhalwal K, PHCOG REV: General Review, J Ethnopharmacology, 2010, 127 (1), 98-102. Pharmacognosy: The changing Scenario, Pharmacog Rev, 24 Abhishek M, Gupta V, Verma SK, Singh SK, Prakash A, 2007, 1 (1), 1-5. Prasad GBKS and Dua VK, Anti-inflammatory activity of ANSARI et al: PHARMACOGNOSTICAL EVALUATION OF ROOT OF GUMMA 95

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