Phytochemical Studies and Quantification of total content of Phenols, Tannins and Flavonoids in selected endangered plant species

 

Kavitha Sagar*, Soma Aneesha, Pooja Uppin, Gowthami

Department of Botany, Vijayanagara Sri Krishnadevaraya University, Ballari, Karnataka.

*Corresponding Author E-mail: kavcsa@gmail.com

 

ABSTRACT:

Four endangered plant species Mimusops elengi, Erythroxylum  monogynum, Holoptelea integrifolia  and Catha edulis were selected to estimate phytoconstituents and quantify total phenols, tannins and flavonoids. Healthy leaves were collected from Mahatma Gandhi Botanical garden and Herbarium, GKVK, University of Agricultural Sciences, Bengaluru and after drying the leaves were subjected for extraction using methanol as solvent and dissolved in 100ml of methanol and stored in air tight bottles for further phytochemical analysis. Total content of phenols, tannins with Gallic acid curve and flavonoids with quercetin by standard methods were estimated. The leaf extract of Mimusops elengi contained maximum amount of tannins and Erythroxylum  monogynum contained minimum amount. Maximum amount of phenols was found in Holoptelea integrifolia. Maximum flavonoids was recorded in Catha edulis. It is further suggested that leaves of  Mimusops elengi, Erythroxylum  monogynum, Holoptelea integrifolia and  Catha edulis prove to potential plants for bioprospection evaluation due high content of phenols, tannins and flavonoids. Simultaneously the conservation of these species must be indicated in our localities, since these are highly important plants and are being exploited due to which these have gained endangered status.

 

KEYWORDS: Endangered, Catha edulis, conservation, flavonoids, phenols, tannin.

 

 


INTRODUCTION:

Plants in general contain important compounds which are helpful to the human welfare and medicinal plants in particular, are the valuable and potent sources of unique phytochemicals which are frequently used in the development of drugs against a wide range of ailments. Large fraction of the world population, especially in the developing and under developed countries still depends mainly on medicinal plants mentioned in the traditional systems.

 

Endangered plant species existing in such small number that it is in danger of becoming extinct especially such a species placed in danger, as a result of the activity one of the principal factor in the endangerment or extinction of the species is the destruction or pollution of its native habitat. In Karnataka there are aproximately 183 endangered plant specieswhich are important in terms of biodiversity assessment26. They play a very important role in environment of the biodiversity of the country.  In this view, these plants must be conserved and also preserved as they have proven medicinal properties evidenced by several reports on ethno botanical and medical uses.

 

 

 

Mimusops elengi L. (Sapotaceae). Used in Ayurvedic medicine in which it is supposed to be astringent cooling, anthelmintic, and tonic. It is mainly used for dental ailments such as bleeding gums, dental caries1. M. elengi contains variety of active phytoconstituents and thus possess various kinds of biological and pharmacological activities. It possess activities like antihyperglycemic2, antihemorrhoidal3, antifungal4,5, antinociceptive and diuretic effects, antiviral, anticariogenic6,7,8, antibacterial5, free radical scavenging9,10, antineoplastic, gastroprotective11 enhancing activity12 and cytotoxic activities.

 

Holoptelea integrifolia (Roxb.) Planch. (Ulmaceae). It is used traditionally for the treatment of inflammation, gastritis, dyspepsia, intestinal worms, vomiting, wound healing, leprosy, diabetes13. Bark and leaves used as bitter, astringent, anti-inflammatory digestive, anthelmintic, urinary astringent14. Chemistry and medicinal uses has been reported15.

 

Catha edulis (Vahl.) Forssk. ex Endl. (Celastraceae). Khat is a high-cash income crop. It is profitable to the huge number of people involved in its production and marketing including farmers, distributors and merchants. In Ethiopia, 85 – 90% of the khat produced is exported16. Phytochemical studies are reported17 by Adugna. Fresh leaves are chewed and consumed as a tea, to achieve a state of Euphoria18 and may also be used by farmers and labourers for reducing physical fatigue or hunger, and students for improving attention19.

 

Erythroxylum monogynum Roxb. (Erythroxylaceae). It is a well-known plant in traditional medicine which is found widely in southern parts of India and in Karnataka (Chikkamagalur, Hassan, Mysore) Kerala (Idukki, Kollam, Thiruvananthapuram) in the southern parts of Andhra Pradesh. Extract of the leaves mixed with yoghurt and is administered to kill intestinal worms, leaf juice is used for treatment of jaundice, and it is taken to cure many disease such as stomachache, dyspepsia, fever and dropsy in Ayurveda medicine.

 

In most hydroxyl group produced by plants mainly for protection against stress, the phenolics play important role in plants. Phenolic compounds are one of the major chemical classes of plants secondary metabolites. They play important role in the defense of plants against pathogens, diseases, parasites and predators. They involve in a number of physiological mechanism such as antioxidant activity produced by a plant depends on several factors such as temperature, UV-light nutrition available to the plant and genetic factors.

 

 

 

 

Flavonoids are a group of plant metabolities thought to provide health benefits through cell signaling pathways and antioxidant effects. The molecules are found in a variety of fruits and vegetables flavonoids are polyphenolic molecules containing 15 carbon atoms and are soluble in water.

 

Tannins (commonly referred to as tannic acid) are water-soluble polyphenols that are present in many plant foods. They have been reported to be responsible for Decreases in feed intake, growth rate, feed efficiency, net metabolizable energy, and protein digestibility in experimental animals. Many tannin molecules have also been shown to reduce the mutagenic activity of a number of mutagens. Many carcinogens and/or mutagens produce oxygen-free radicals for interaction with cellular macromolecules. The anticarcinogenic and antimutagenic potentials of tannins may be related to their antioxidative property, which is important in protecting cellular oxidative damage, including lipid peroxidation. The generation of superoxide radicals was reported to be inhibited by tannins and related compounds. The antimicrobial activities of tannins are well documented. The growth of many fungi, yeasts, bacteria, and viruses was inhibited by tannins. The antimicrobial property of tannic acid can also be used in food processing to increase the shelf-life of certain foods, such as catfish fillets20.

 

In view of above, the present study was conducted to determine the phytochemicals and total phenols, flavonoids and tannins in the above mentioned endangered species.

 

METHODS AND MATERIALS:

Sample collection:

The plant material was collected from Mahatma Gandhi Botanical garden and Herbarium, GKVK, University of Agricultural Sciences, Bengaluru. The collected plants are washed with water to remove the dust particles and dried under shade.

 

Preparation of Plants extract:

The dried leaves of different plants were ground separately to make a powder sample by using Grinder. The obtained powder samples were packed in a sealed plastic covers or air tight bottles. 20 g of each powder sample was dissolved in 100ml of methanol in 250ml conical flask, covered with aluminium foil and kept in mechanical shaker for 24hours. Later the extract was filtered and solvent was evaporated in petriplates /watch glasses for 2days. Then dried extract was carefully collected by dissolving in 100ml methanol in conical flask and kept in refrigerator for further phytochemical analysis. Phytochemical analysis was done following standard methods`21.

 

i. Quantification of total phenols by Folin-Ciocalteu method:

Total phenol was determined by Folin-Ciocalteu reagent method with modification. From each crude extracts (1 mg) was dissolved in methanol (1 ml). A total of 10% Folin -Ciocalteu reagent was prepared by adding Folin-Ciocalteu reagent (10 ml) in water (90 ml). Then, 5% Na2Co3 (3 g) in water (50ml). Each crude sample (200 µl) was taken in a test tube and added 10% Folin - Ciocalteu reagent (1.5ml). Then all test tubes were kept in a dark place for 5 min. Finally 5% Na2Co3 (1.5 ml) was added to the solution and mixed well by hand. Again all the test tubes were kept in the dark for 2h. The absorbance was measured for all solution by using UV-spectrophotometer at constant wavelength 750nm.

 

Gallic acid standard curve:

A standard curve was prepared using Gallic acid. For this, 10mg Gallic acid was dissolved in 100% methanol. Several dilutions of Gallic acid methanol were prepared, viz. 50, 100, 150, 200, 250µg/ml. One ml aliquot of each dilution was taken in a test tube and diluted with 9ml of distill water and this 2.5ml Folin-Ciocalteu’s reagent was added. This was followed by the addition of 2.5ml of 7.5% NaHCO3 in each test tube. The resulting mixture was left to stand for 30 minutes at room temperature. Absorbance of the standard was measured at 765nm using UV spectrophotometer against blank.

 

Quantification was done on the basis of a standard curve of Gallic acid. Based on the measured concentration of phenol was read (mg/ml) from the calibration line; then the content of phenol in extracts was expressed in terms of Gallic acid equivalent (mg of GA/g of extract).

 

Gallic acid equivalent (GAE) T = C × V/m, where GAE is the Gallic acid equivalence (mg/ml); V is the volume extract (ml) and M is the wfour (g) of the pure plant extract.

 

The % yield extracts was calculated by using the following formula:

 

  Weight of extract obtained (g)

% yield=-------------------------------------×100

  Weight of plant material (g)

 

 

 

ii. Quantification of total content of flavonoids:

The total flavonoids were determined according to the method described by Djeridane et al., (2006). One ml of extract (0.5 mg/mI) was mixed with 1ml Aluminium chloride (2%). The mixture stirred and kept at room temperature for 15min. The absorbance was measured at 430nm using a HACH DR/4000 UV spectrophotometer.

 

The total flavonoids were reported as mg routine equivalents (RE) per g dry weight (DW).

Total flavonoid content was calculated with standard curve equation and the formula

 

Total flavonoid content (% w/w)

= RE×V×D×10-6 ×100 /W

Where, RE – Rutin equivalent (µg/ml); V – Total volume of sample (ml); D – Dilution factor; W – Weight of sample (gm).

 

iii. Quantification of total content of Tannins:

Total tannins were estimated by Folin-Denis Method. The intensity is measured in a spectrophotometer at 700 nm. Add 1 ml of the sample extract to a 100-ml volumetric flask containing 75 ml water. Add 5 ml of Folin-Denis reagent, 10 ml of sodium carbonate solution and dilute to 100 ml with water. Shake well and read the absorbance at 700 nm after 30 min. Prepare a blank with water instead of the sample. A standard graph was prepared by using 0–100 mg tannic acid. The tannin content of the samples was calculated as tannic acid equivalents from the standard graph.

 

RESULT:

Preliminary quantitative analysis of selected endangered species:

The presence or absence of phytochemical compounds are represented by positive or negative signs as represented in Table 1.

 

Quantification of total phenols:

The present showed the presence of maximum content of total phenols in Holoptelea integrifolia (Fig: 2). The phenols and tannins content was estimated by Gallic acid standard (Fig-1) and expressed in milligram of Gallic acid equivalent (GAE) in different concentration viz 50, 100, 150, 200, 250mg/ml (Fig. 2).

 

 

 

 


Table-1: Preliminary quantitative analysis of selected endangered species

Plant species

Proteins

Phenol and Tannins

Saponins

Glycosides

Carbohydrates

Alkaloid

Flavonoids

M. elengi

_

+

+

+

_

_

_

C. edulis

_

+

+

+

+

_

_

H. integrifolia

_

+

+

+

-

NA

+

 E. monogynum

 +

 +

-

 +

 -

 NA

-

 


 

 

Fig- 1: Standard Gallic acid Curve.

 

 

Fig.2: Total Phenols

 

Quantification of tannins:

Tannin is one of the major active ingredients found in plant based medicines. It serves as caustics for cationic dyes used in dyes stuff industry as well as in the production of inks and also used for wine, fruit juice and beer classification in food industries22. Production of coagulant in rubber production23 as well as they possess antiviral antibacterial and antitumor activity. The study showed the presence of maximum content of total tannins in Mimusops elengi and minimum in Erythoxylum monogynum (Fig. 3).

 

 

Fig. 3: Total tannins

Quantification of flavonoids:

The leaf extracts of four endangered plant species showed maximum flavonoid contents in Catha edulis and minimum in Holoptelea integrifolia (Fig. 4 and 5). The total contents of flavonoids was determined24 by Chang et al., method.

 

 

Figure- 4: Standard Calibration of Quercetin

 

 

Fig. 5: Total Flavonoids

 

The flavonoids as known to have antioxidant effects and have been shown to inhibit the initiation, promotion and progression of tumor and reduction of coronary heart disease has been reported to be associated with intake of flavonoid from the antioxidant properties of flavonids and other biological functions include protection against platelet aggregation micro organism hepatotoxin, viruses, tumor free radicals, inflammation and allergy.

 

In the present investigation showed that of all four plant extracts were positive for quantitative phytochemical extraction except few. And also total phenols, tannins and flavonoids were quantified and they were present in less quantity depending upon the concentration of plant extracts. These phytocompounds are involved in anti-biofilm activity, anti-bacterial and antifungal activity as evidenced by our work.

DISCUSSION:

Plants have great potential for producing new drugs for human benefit. The increased interest in plant drugs is mainly because of wide spread belief that” Herbal medicine” is safer than synthetic drugs having no side effects. The plants have good antioxidant ability that can be attributed to the phenolic acids, flavonoids and tannins found in the plant. The chemical constituents in the plants or crude extracts are known to be biologically active ingredients. Some chemical constituents are directly responsible for different activity such as antioxidant, antimicrobial, antifungal and anticancer. All these secondary metabolites components showed antioxidant and antimicrobial properties through different mechanism.

 

In the present study, the leaf extract of Mimusops elengi contain maximum amount of Tannins and minimum in Erythroxylum monogynum. Holoptelea integrifolia contains maximum amount of phenols. This study shows the maximum content of flavonoids in Catha edulis, but when compared to other reports Catha edulis contain minimum level of flavonoids. Our study shows the minimum level of flavonoids in Holoptelea integrifolia, when compared to other reports shows little variations25.

 It is further suggested that leaves of Mimusops elengi, Erythroxylum monogynum, Holoptelea integrifolia and Catha edulis prove to be potential plants for bio prospection evaluation due high content of phenols, tannins and flavonoids. Simultaneously the conservation of these species must be indicated in our localities, since these are highly important plants and are being exploited due to which these have gained endangered status.

 

CONFLICTS OF INTEREST:

The authors declare no conflict of interest.

 

REFERENCES:

1.     S. N. Yoganarasimhan. Medicinal Plants of India. Interline Publishing Pvt. Ltd., Bangalore (1996); p. 88: Vol. 1.

2.     Hanumanthachar J, Milind P. Evaluation of the memory and learning improving effects of Mimusops elengi in Mice. Int J Drug Disc Herbal Res. 2011; 1(4):185–192.

3.     Bharat Gami. Evaluation of pharmacognostic and antihemorrhoidal properties of Mimusops elengi Linn. Ph.D. Thesis. 2007. Veer Narmad South Gujarat University.

4.     Manjeshwar SB, Ramakrishna JP, Harshith PB, Princy LP, Rekha B. Chemistry and medicinal properties of the Bakul (Mimusops elengi Linn): A review. Food Res Int. 2011; 44 (7):1823–1829.

5.     Prabhat A, Navneet, Avnish C. Evaluation of antimicrobial activity of six medicinal plants against dental pathogens. Report Opinion. 2010; 2(6): 37–42.

6.     Shah PJ, Gandhi MS, Goswami SS, Santani D. Study of Mimusops elengi bark in experimental gastric ulcers.  J Ethanopharmacol.  2003; 89(2-3):305–311.

7.     Kusumoto IT, Nakabayashi T, Kida H, Miyashiro H, Hattori M, Namba T. Screening of various plant extracts used in Ayervedic medicine for inhibitory effects on human immunodeficiency virus thpe 1 (HIV-1) protease. Phytother Res. 1995; 9:180–184.

8.     Kala S, Johnson M, Iyan R, Dorin B, Jeeva S, Janakiraman N. Preliminary phytochemical analysis of some selected medicinal plants of south India. J Natura Conscientia. 2011; 2(5):478–481.

9.     Sahaa MR, Hasana SMR, Aktera R, Hossaina MM, Alamb MS, Alam MA, et al. In vitro free radical scavenging activity of methanol extract of the leaves of Mimusops elengi linn. Bangl. J Vet Med. 2008;6(2):197–202.

10.  Nithya N, Rohini S, Arun D, Balakrishnan KP. Antityrosinase and antioxidant activities of various parts of Mimusops elengi: a comparative study. Int J Res Cosm Sci. 2011; 1(1):17–22.

11.  Dabadi P, Koti BC, Vijay T, Chandrakala, Manjuntha SK. Antiulcer activity of Mimusops elengi bark extracts against serotonin induced ulcer in rats. Int Res J Pharm. 2011; 2(8):173–176.

12.  Hadaginhal RV, Tikare VP, Patil KS, Bhanushali MS, Desai NS, Karigar A. Evaluation of cognitive enhancing activity of Mimusops elengi Linn on albino rats. Int J Res in Aur and Pharm. 2010; 1(2): 484–492

13.  PK Warrier, VPK Nambiar and C Ramakutty. Indian Medicinal Plants: A Compendium of 500 Species. Orient Longman, 1995.

14.  ND Prajapati, SS Purohit and AK Sharma. A Handbook of Medicinal Plants: A Complete Source Book, Agrobias, Jodhpur, India, 2003.

15.  Harleen Kaur Sandhar, Mohanjit Kaur, Prashant Tiwari, Manoj Salhan, Pardeep Sharma, Sunil Prasher, Bimlesh Kumar. Chemistry and Medicinal properties of Holoptelea Integrifolia. International Journal of Drug Development and Research. Jan- March 2011; 3(1): 6-11.

16.  Lemessa D (2001) Khat (Catha edulis): botany, distribution, cultivation, usage and economics in Ethiopia. Addis Ababa: UNEmergencies Unit for Ethiopia.

17.  Adugna Y 2009. Phytochemical studies on khat (Catha edulis) M.Sc. Thesis. Addis Ababa University, Ethiopia.

18.  https://www.ncbi.nlm.nih.gov/pmc/articles

19.  https://en.m.wikipidia.org

20.  Chung KT, Wong TY, Wei CI, Huang YW, Lin Y. Tannins and human health: a review. Critical reviews in food science and nutrition. 1998; 38(6): 421-464.

21.  Sadashivam S and Manickam A. Biochemical methods. New age International publications. 1996.

22.  Würdig G, Woller R. Chemie des Weines. Stuttgart, Eugen Ulmer GmbH and Co., 1989; 926s.

23.  J Falbe and M Regitz, CD RÖMPP Chemie Lexikon, Version 1.0, Georg Thieme Verlag, Stuttgart/New York, 1995.

24.  Chang C, Yang M, Wen H, Chern J. Estimation of total flavonoid content in propolis by two complementary colorimetric methods. Journal of Food and Drug Analysis. 2002; 178-182.

25.  Chukwama Ezeonu and Chigozie Ejikeme.. Qualitative and Quantitative Determination of Phytochemical Contents of Indigenous Nigerian Softwoods. 2016. New Journal of Science.

26.  http :// www. Indiasendangered.com

 

 

 

 

Received on 18.06.2018          Modified on 28.06.2018

Accepted on 11.07.2018  ©A&V Publications All right reserved

Res. J. Pharmacognosy and Phytochem. 2018; 10(4): 277-281.

DOI: 10.5958/0975-4385.2018.00044.4