COMPARATIVE STUDY ON PHARMACOGNOSTIC AND PHYTOCHEMICAL COMPOSITION OF SEED COAT AND COTYLEDON OF CAJANUS CAJAN L.HTML Full Text
COMPARATIVE STUDY ON PHARMACOGNOSTIC AND PHYTOCHEMICAL COMPOSITION OF SEED COAT AND COTYLEDON OF CAJANUS CAJAN L.
H. Pratima * 1 and Pratima Mathad 2
Department of Post-Graduate Studies and Research in Botany 1, Karnataka State Women’s University, Vijayapur - 568101, Karnataka, India
Department of Post - Graduate Studies and Research in Botany 2, Gulbarga University, Kalburgi - 585106, Karnataka, India.
ABSTRACT: Cjanus cajan (L.) Millsp belonging to the family Fabaceae commonly known as pigeonpea or arhar. The present study conducted to assess the phytochemical and pharmacognostic in terms of organoleptic, fluorescence analysis and physicochemical parameters of seed coat and cotyledon of C. cajan. Differential extraction yielded of seed coat extracts of petether, chloroform, ethanol and aqueous shows 6.3%, 1.14%, 11.36% and 9.32% and cotyledon extracts of pet ether, chloroform, ethanol and aqueous shows 7.34 %, 4.25%, 10.45 % and 8.45 % respectively. Physicochemical parameters such as total ash, acid insoluble ash and water soluble ash of seed coat and cotyledon were found to be 14.5% and 11.5%, 8.35% and 7.36%, 2.11% and 3.39% respectively. Further the mineral elements like zinc, magnesium and copper content were found more in seed coat whereas, maximum content of iron were observed in cotyledon. The fluorescent analyses of powdered drug play an important role in the determination of quality and purity of the drug. Phytochemical analysis revealed the presence of glycosides, tannins, flavonoids, phenols, lignins, alkaloids, steroids and glycosides in seed coat and cotyledon. The maximum content of phenols, flavonoids, tannins, lignins, alkaloids, steroids were found in seed coat compared to cotyledon, whereas glycosides content was more in cotyledon. The pharmacognostic study revealed the purity of the sample and helps to differentiate the plant sample from the adulterants.
Cajanus cajan, Cotyledon, Pharmacognostic, Phytochemical, Seed coat
INTRODUCTION: The dietary plants are also considered to be medicinally which are going to reduce the risk of several chronic diseases. In recent years a lot of research has been done in the development of chemopreventive agents derived from foods that constitute integral parts of the human diets 1.
Legumes, which play a crucial role in many diets worldwide are thought to be related with beneficial health implications in chronic diseases such as certain cancer types (colon, breast, prostate) 2 and diabetes 3. Except from their known high nutritive value, significant quantities of phytochemical compounds are identified in legumes and considered to be responsible for their beneficial effects 4. Cajanus cajan (L.) Mill sp (In english: red gram, hindi: tur or arhar, kannad: togari, sanskrit: adhaki) is a perennial member of the family Fabaceae. Other common names are pigeon pea, congo pea, gungo pea and no-eye pea 5.
Among the “Pigeon pea” is the globally popular name that was coined by Plukenet 6 in Barbados, where the crop was in barren lands for feeding its seeds to pigeons. It is originated in Asia and is being cultivated over 3000 years. C. cajan is grown throughout the tropical and subtropical countries especially in South Asia, Eastern and southern Africa, Latin America and Australia. About 90% of the world production of C. cajan is contributed by India where it is cultivated in semi- arid regions as kharif. It cultivated mainly Karnataka, Maharastra, Tamilnadu, Rjasthan states. In Karnataka C. cajan is grown in an area of 5.83 lakh hectares with a production of 2.57 lakh tonnes. It is largely grown in the northern parts of the state especially in Kalaburgi district, which is called “Pulse bowl of Karnataka”. C. cajan is very prominent crop in this district and covers approximately an area of 2.5 lakh hectares occupying 65% of the total area under C. cajan cultivation in Karnataka state 7. Besides its high nutritional value, C. cajan is also used as traditional folk medicine in India, China, Philippines and some other nations. The seeds are astringent, acrid, sweet, cooling, anthelmintic, resolvent, expectorant and constipation 8. Further, the extract from seeds showed hypolipidemic, antioxidant and antimicrobial activities 9.
The pharmacognostic studies of leaf, stem, root and seeds of C. cajan have been reported by other workers 10, 11, 12, 13. The C. cajan seed coat and cotyledon proper and detailed pharmacognostical studies have not been reported so far. So an attempt was made to standardize the drug on the basis of parmacognostic and phytochemical parameters. The objective of the present study to evaluate various pharmacognostical standards like organoleptic, ash values, mineral contents fluorescence analysis and phytochemical analysis of C. cajan seed coat and cotyledon.
MATERIAL AND METHODS:
Plant collection: The seeds of Cajanus cajan L. varieties Maruti (ICP-8863) were collected from the field of Gulbarga District in Karnataka. Seeds were moistened for 1h and then dried in oven at 550C overnight. The hull or seed coat was removed mechanically means by using hand grinder. The two fractions like seed coat and cotyledon were made into fine powder using grinder.
Organoleptic studies of crude extract: For the determination of organoleptic characters such as color, nature, taste and yield of the extracts, 100g of dried and powdered plant material was successively extracted in the Soxhlet extractor using petroleum ether, chloroform, ethanol (95%, v/v) and distilled water solvents in the increasing order of polarity for 18h. The resulting liquid extracts were evaporated to dryness under reduced pressure. The yield of the extracts were calculated using the following formula 14, 15.
Determination of total ash and acid insoluble ash content: Two grams of dried and powdered plant material was taken in the pre-weighed clean sintered silica crucibles. Then, they were incarnated by gradual increasing of the temperature (400-5000C) in the muffle furnace for 6h. The crucible were cooled to room temperature in a desecrator and the weight of ash content was weighed in as electronic digital balance (Anamed make).
The total ash content of the plant material thus obtained was boiled for 15min, after adding 25ml of 25 %( v/v) HCl and was allowed to cool. It was filtered through a Whatman filter paper No. 44 (ash less). The insoluble ash thus retained on filter paper along with paper was ignited in a pre-weighed sintered crucible (1000C). Then the crucible along with the residue was weighed and calculated the total ash and acid insoluble ash content using the following formula 14.
Total ash content (%) = Z-X × 100
Where, Z= Weight of the crucible; X = Weight of the crucible with ash; Y = Weight of the plant material taken (g).
Estimation of minerals: Estimation of minerals element by the method of AAS (Atomic Absorption Spectrometer) 16. Five gram of seed coat and cotyledon ash was dissolved in 25ml of dilute HCl (1:1v/v) in the china dish and covered with a watch glass and incubated 20-25 minute on hot water bath, gradually cooled and filtered through a whatman filter paper No.44 (ashless).
10ml of filtrate was taken in 100ml volumentric flask and made its final volume to 100ml by adding distilled water. This solution was diluted to 10 times using distilled water, this stock ash solutions were used for the detection and estimation of elements using Atomic Absorption Spectrometer. The mineral content of the sample was calculated using the following formula
Mineral content (ppm) = Z×S×A
Where, Z= Mineral content of the standard solution, S=Reading of the test sample of a mineral, Y= Reading of the standard solution of the mineral, A= Dilution factor, W= weight of plant material (g), 100= volume of the stock solution.
Fluorescent studies powder drugs: A pinch of dried and powdered plant material was taken in a clean test tube with about 10ml of solvent like acetone, benzene, petroleum ether, chloroform, ethanol, glacial acetic acid, HCl, HNO3, methanol and distilled water. All the tubes were shaken well and incubated for about 30min. The colors of the drug solutions thus obtained were observed for their characteristic color reaction under the visible light (fluorescent tube) and ultra violet light (UV366nm) and were recorded by comparing with a standard color chart 17.
Qualitative Phytochemical test: Qualitative phytochemical analysis was carried out on the extracts (viz, petroleum ether, chloroform, alcohol, and aqueous) to determine the presence or absence of metabolites such as alkaloids, flavonoids, glycosids, saponins, tannins, phenols, lignins, steroids and saponins as described by Harborne 18.
Quantitative phytochemical analysis: The quantitative estimation of secondary metabolites such as phenols was estimated by the method of Malick and Singh 19, flavonoids content was estimated by the method of Jia et al. 20, the alkaloid was estimated by the method of Harborne 21, the total glycoside was estimated according to the method of Huguchi and Hanssen 22, the total steroids were estimated according to the method of Scanchez et al. 23. Further, the total tannins were estimated by Folin Denis method 24 and the total lignins were estimated according to the method of Stafford 25.
Statistical analysis: The data of all measurements are means from three replications. Data and statistical significance of difference were evaluated with analysis of variance (ANOVA) using SPSS 10.0 package
RESULTS AND DISCUSSION:
Organoleptic studies: The organoleptic characters such as extractive values, colour, nature, taste of the crude drugs of different parts of C. cajan and their results are presented in Table 1.The extraction yield of seed coat of different solvents varied from 5.14% to 11.36% and could be ranked from high to low i.e., ethanol > aqueous > pet ether > chloroform. Similarly, the yield of cotyledon extracts were 4.25% to 10.45% and could be ranked from high to low i.e., ethanol > aqueous > pet ether > chloroform respectively. The maximum extraction yield was observed in seed coat compared to cotyledon. The results revealed that ethanol shows higher extractive values may be its unique feature of dissolving all polar and nearly all new polar constituents. Similarly, ethanol extract shows higher extractive value in croton seed 26. The crude extracts of seed coat and cotyledon of C. cajan have exhibited a wide range of colour.
The pet-ether extracts shows dark brown colour in seed coat and yellowish in cotyledon, whereas, chloroform extracts shows dark brown colour in seed coat and yellowish in cotyledon respectively. The ethanolic extracts were dark brown colour in seed coat and golden yellow colour in cotyledon. Whereas, the aqueous extract of seed coat and cotyledons were brown in colour. Further, the extracts of C. cajan seed coat and cotyledon have either pungent bitter or bitter in taste. Of these, the pet-ether and chloroform extracts were pungent bitter while the remaining was bitter in taste. Similarly, the nature of these extracts varies from sticky (pet-ether), waxy (chloroform), resin (ethanol) and powdery (aqueous) respectively.
Total Ash, Acid insoluble ash and Water soluble ash: In the present investigation the total ash and acid insoluble ash value of seed coat and cotyledon of C. cajan were recorded in Table 2. The highest content of total ash 14.5%, acid insoluble ash 8.35% and water soluble ash 2.11% were recorded in seed coat followed by cotyledon, which had 11.5% of total ash 7.36% of acid insoluble ash 3.39% of water soluble ash respectively. Similarly, the 3.70% of total ash have been reported in stalk of C. Cajan 27. The variation in the percent of ash is probably due to the nature of metabolites they possess. The ash value is useful in determining authenticity and purity of the drug and also these values are important quantitative standards.
TABLE 1: ORGANOLEPTIC CHARECTERS OF SEED COAT AND COTYLEDON EXTRACTS OF C. CAJAN
|Characters||Successive extracts||Plant parts|
|Taste||Pet-ether||Pungent Bitter||Pungent Bitter|
|Chloroform||Pungent Bitter||Pungent Bitter|
|Aqueous||Amorphous Powder||Amorphous Powder|
TABLE 2: TOTAL ASH, ACID INSOLUBLE ASH AND WATER SOLUBLE ASH VALUE OF C. CAJAN
|Physical constant||Plant parts|
|Acid insoluble ash||8.35%||7.36%|
|Water soluble ash||2.11%||3.39%|
Mineral Elements of C. cajan: Results of element analysis of seed coat and cotyledon by AAS (Atomic absorption spectrophotometer) are recorded in Table 3. The highest content of zinc (0.0244±0.0012ppm), magnesium (0.0230 ±0.0131 ppm) and copper (0.1811±0.0511ppm) were recorded in seed coat compared to cotyledon whereas; the maximum amount of iron (0.0247±0.0121ppm) was recorded in cotyledon compared to seed coat. The very negligible amount of lead was detected in cotyledon (0.0001±0.0001 ppm) followed by seed coat (0.0003±0.0002ppm). Similarly, C. cajan. leaves contain zinc, iron magnesium, calcium and copper 28, 29.
TABLE 3: MINERAL ELEMENTS OF SEED COAT AND COTYLEDON OF C. CAJAN
|Iron (ppm)||0.0134 ±0.0034||0.0247±0.0121|
|Magnesium (ppm)||0.0230 ±0.0131||0.1011±0.0123|
Note: ND-Not detected
Each value is expressed as mean ± S.D (n=3) and statistically significant at P<0.05
Fluorescent studies of C. cajan: The fluorescent study of dried powder of seed coat and cotyledon of C. cajan were observed in both under the visible and ultra-violets (UV) light by treating with various solvents showed characteristic colours were recorded in the Table 4. Among various solvents tested acetone, hydrochloric acid, petroleum ether did not show any fluorescence in both seed coat and cotyledon powder. Whereas, distilled water, methanol, acetone, chloroform, glacial acetic acid, ethanol, sulphuric acid, nitric acid showed characteristic colouration in both seed coat and cotyledon powder. These results are supportive with fluorescent studies performed with Lens culinaris seed coat and cotyledon powders 30. The C.cajan leaf and seed powder showed characteristic colouration in distilled water, benzene, chloroform, HCl, H2SO4, HNO3, CH3COOH, 5% FeCl3, 5% I2, 1N NaOH, picric acid and 1N NaOH+methanol solvents 13.
TABLE 4: FLUORECENT STUDIES OF SEED COAT AND COTYLEDON POWDERS OF C. CAJAN
|Sl. No.||Treatment||Seed coat||Cotyledon|
|1||Distilled water||Light Yellow||Brown||Yellowish||Light Yellow|
|2||Ethanol||Light Yellow||`Colourless||Colourless||Light Yellow|
|3||Methanol||Light yellow||Colourless||Colourless||Light Yellow|
|5||Chloroform||Light yellow||Brown||Colourless||Light Yellow|
|6||Glacial acetic acid||Light Yellow||Light Brown||Colourless||Light Yellow|
|7||Sulphuric acid||Light Yellow||Brown||Brownish||Light Brown|
|8||Nitric acid||Yellowish||Light Brown||Yellowish||Light Yellow|
Preliminary screening of phytochemicals: The phytochemicals detected in C. cajan L. seed coat and cotyledon extracts are listed in Table 5. Test for flavonoid, tannins, phenols, glycosides, alkaloids, steroids and lignins were positive in all extracts except pet ether and chloroform extract alkaloids and steroids are not detected in both seed coat and cotyledon. The saponine is absent in all extracts of seed coat and cotyledon. Similarly, C. cajan seed extracts showed the presence of alkaloids, glycosides, resins, phenols, steroids, lignins, fats and oils in acetone, aqueous, benzene, chloroform, diethyl ether, methanol extracts in varying quantities 13. The alcohol and water extracts of Cicer arietinum seeds shows the presence of phytosterols, flavonoids, tannins, phenolic compounds 31.
TABLE 5: PHYTOCHEMICAL SECREENING OF EXTRACTS OF SEED COAT AND COTYLEDON OF C. CAJAN
|Secondary metabolites||Name of the test||Seed coat||Cotyledon|
|Ellagic acid test||+||+||+||+||+||+||+||+|
|Conc. H2SO4 test||+||+||+||+||+||+||+||+|
‘+’: Present, ‘-‘: Absent; PE: Pet ether, CHCl3: Chloroform, Et-OH: Ethanol, Aq: Aqueous extract
Phytochemical constituents: The results shows that seed coat contained higher amount of phenols (3.15±0.85 mg/100 mg), flavonoids (290.13±0.62 mg/100), tannins (190.0±0.98 mg/100 mg), lignins (200.0±0.82 mg/100 mg), alkaloids (0.82±0.12 mg/100 mg) and steroids (0.63±0.05 mg/100 mg) whereas, the maximum amount of glycosides (115.34 ±0.81 mg/100 mg) was observed in cotyledon (Table 6). Phenolic compounds which inhibit the activities of α- amylase, α- glucosidase and protease, provide an attractive target for the development of potential therapeutic agents to treat diabetes, pancreatitis, coagulation and neoplastic diseases 32. The C. cajan leaves crude extracts are high contents of phenol and flavonoids and are traditionally used in treatment of jaundice and diabetes 33. The highest value of tannins was observed in seed coat which serves as astringent properties for healing of wound, inflaming mucous membrane and source of antioxidant 34. The results shows that highest value of lignin 200.0±0.82 mg/100 mg was observed in seed coat compared to cotyledon. Similarly, brown cow pea seed coat shows 236.0 gm Kg-1 lignins 35 and lignins also act as antioxidants 36. The seed coat contains high amounts of these bioactive compounds; it is reliable to possess large number of medicinal values.
TABLE 6: CONSTITUENTS OF SECODARY METABOLITES IN SEED COAT AND COTYLEDON OF C. CAJAN
Each value is expressed as mean ± S.D (n=3) and statistically significant at P<0.05
CONCLUSION: The present study on Pharmacognostical and Phytochemical evaluation of C. cajan seed coat and cotyledon will provide useful information for its identification and authentication of the drug in future. Phytochemical studies revealed that presence of phenols, flavonoids, alkaloids, steroids, tannins, lignins and glycosides. The outcome of this phytochemical screening thus suggest that seed coat are rich source of secondary metabolites are highly recommended in everyday diet of man and health benefits.
ACKNOWLEDGEMENT: The authors are thankful to the Chairman, Department of Botany, Gulbarga University Gulbarga and the Principal, Karnatak College, Bidar for providing the laboratory facilities.
CONFLICT OF INTEREST: The authors of this article declare that we have no conflict of interest in this study.
- Shukla Y, Kumar Pal S: Dietary cancer chemoprevention: an overview. Hum Genet 2004; 4: 265-276.
- Mathers JC: Pulses and carcinogenesis: potential for the prevention of colon, breast and other cancers. J. Nutr 2002; 88: 273–279.
- Venn BJ, Mann JI: Cereal grains, legumes and diabetes. J. Clin. Nutr 2004; 58: 1443–1461.
- Rochfort S, Panozzo J: Phytochemicals for health, the role of pulses. Agric. Food Chem. 2007; 55: 7981–7994.
- Ambasta SP: The useful plants of India 4th New Delhi: National Institute of Science Communication 2004: 94–95.
- Plukenet L: Phytographia
- Sidram : Analysis of Organic Farming Practices in Pigeonpea in Gulbarga District of Karnataka State. Thesis Department of Agricultural Extension Education, College of Agricultural Science Dharwad,Karnataka, India 2008.
- Narayan BSV, Gatzoulis MA, Kilner PJ: Non-invasive imaging in adult congential heart disease using cardiovascular magnetic resonance. Cardiovascular Medicine 2007; 1(8): 23-29.
- Oboh G: Antioxidant properties of some commonly consumed and underutilized tropical legumes. European Food Research and Technology 2006; 224 (1): 61-65.
- Mahitha B, Archana P, MD Ebrahimzadeh H, Srikanth K, Rajinikanth M, and Ramaswamy N: In vitro antioxidant and pharmacognostic studies of leaf extracts of Cajanus cajan (L.) Millsp. Indian journal of Pharamaceutical Sciences 2015: 172-179
- Mahitha B, Archana P, Srikanth K, Ramaswamy N: Pharmacognostical and preliminary phytochemical investigations in stem and root of Cajanus cajan (L) millsp. Res J Biotech 2014; 9(7):36-42.
- Pratima H and Pratima Mathad: Pharmacognostic evaluation and phytochemical analysis of leaves of Cajanus cajan, J. Advances in Developmental Research 2011; 2(2): 181-185.
- Rama Swamy Nanna, Mahitha Banala, Archana pamulaparthi, Archana Kurra, Srokanth kagithoju: Evaluation of phytochemicals and fluorescent analysis of seed and leaf extracts of cajan. Int. J. Pharm. Sci. Rev. Res 2013; 22(1): 11- 18.
- Raghunathan K: Pharmacopoeial standards for Ayurvedic formulations. Central council for Research in Indian Medicine and Homeopathy, E-25, Defense colony, New Delhi 1976:378
- Usha S, Joshi P and Sharma HP: Pharmacognostic studies on Atemisia scoparia Waldst and Kit. Proceedings of Indian Academy of Science (Plant Science) 1984; 93(2): 151-164.
- Chase CR and Pratt R J: Fluorescence of powdered vegetables drugs. Indian J. Exp. Biol. 1949; 33(6):428-432.
- Swhney SK and Sing R: Introductory practical Biochemistry, published by Narosa publishing House, New Delhi 2002: 81-90.
- Harborne JB: Phytochemical methods, A guide to modern techniques of plant analysis 3rd Edn. Chapman and Hall, Madras 1998: 302.
- Malick CP and Singh MB: In plant enzymology and histoenzymology. Kalyani Publishers, New Delhi 1980: 286.
- Jia Z, Tang M. and Wu J: The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals Food Chem 1999; 64: 555-559.
- Harborne JB: The terpenoids In: Phytochemical methods. Chapman and Hall, London 1973; 3:
- Huguchi Takeru and Einar Brochmann-Hanssen: Pharmaceutical analysis, Ist edition, CBS publishers and distributors, New Delhi 1997: 57-58.
- Sanchez-fernandez R, Fricker M, Corben LB, White NS, Leaver CJ, Van Montagu M, Inze D and May MJ: Cell proliferation and hair tip growth in the Arabidopsis root are under mechanistically different forms of redox control. Nat. Acad. Sci. USA 1997; 94: 2745-2750.
- Schanderi SH: In: Method in Food Analysis Academic Press, New York 1970; P. 709.
- Stafford HA: Differences between lignin like polymer formed by peroxidation of eugenol and ferulic acid in leaf section of Phleum. Plant Physiology 1960; 35 (1):108-114.
- Jyoti MG, Vijay VN, Baragi Umapati C, Baragi Pramod C: Pharmacognostic, phytochemical and physicochemical investigation of Croton tiglium International Journal of Pharmacy 2014; 4(3): 140-145.
- Osman Taha Elzaki, Tarig Osman Khider and Safaa Hassan Omer: Pulp and papermaking characteristics of cajan stem from Sudan. American-Eurasian J. Agric and Environ Sci. 2012; 12 (2): 159-163.
- Lawal Opeyemi Uwangbooje: The mineral and phytochemical analysis of the leaves of Senna alata and Cajanus cajan and their medicinal value. International J. Biology, Pharmacy and Allied Sciences 2012; 1(1): 1-11.
- Vidhya Anandhi T and Ahmed John S: Screening of trace metals and secondary metabolites from Cajanus Cajan (L.) Mill sp. And study their antimicrobial efficacy. World J.Pharmaceutical Research 2016; 5(4): 1059-1071.
- Kripi Vohra1, Vivek Kumar Gupta; Pharmacognostic evaluation of Lens culinaris Medikus seeds Asian Pacific Journal of Tropical Biomedicine 2012; 1221-1226.
- Mamta Arora, Satnam Singh and Parneet Kaur: Pharmacognostic and phytochemical evaluation of selected seeds of Cicer arietinum Seeds from Roopnagar Punjab. International journal of pharmaceutical science invention 2013; 2(11): 18-19.
- Kripi Vohra1, Vivek Kumar Gupta; Pharmacognostic evaluation of Lens culinaris Medikus seeds Asian Pacific Journal of Tropical Biomedicine 2012; 1221-1226.
- Asma Hamood Al-Saeedi, Mohammad Amzad Hossain: Evaluation of total phenols, total flavonoids and antioxidant activity of the leaves crude extracts of locally grown pigeon pea traditionally used in Sultanate of Oman for the treatment of jaundice and diabetes.Journal of Coastal Life Medicine 2015; 3(4): 317-321
- Frecer V, Ho B, Ding JL: Interpretations of biological activity data of bacterial endotoxins by simple molecular models of mechanism of action. J. Biochem 2000; 267: 837-852.
- Okwu DE: Phytochemical and vitamin content of indigenous species of south Eastern Nigeria. Sustain Agric. Environ 2004; 6:30-34.
- Mbagwu FN, Okafor VU and Ekeanyanwu J: Phytochemical screening of four edible legumes (Vigna subterranean, Glycine max, Arachis hypogeal and Vigna ungucuilata) found in Eastern Nigeria. Afr. J. Plant Sci 2011; 5: 370-372.
How to cite this article:
Pratima H and Mathad P: Comparative study on pharmacognostic and phytochemical composition of seed coat and cotyledon of Cajanus cajan L. Int J Pharm Sci Res 2017; 8(4): 1751-57.doi: 10.13040/IJPSR.0975-8232.8(4).1751-57.
All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.
H. Pratima * and Pratima Mathad
Department of Post-Graduate Studies and Research in Botany, Karnataka State Women’s University, Vijayapur - 568101, Karnataka, India.
16 September, 2016
16 December, 2016
20 March, 2017
01 April, 2017