IN VITRO QUALITATIVE AND QUANTITATIVE PHYTOCHEMICAL ANALYSIS OF ETHANOLIC AND 50% ETHANOLIC EXTRACTS OF TINOSPORA CORDIFOLIA, MOMORDICA CHARANTIA, CUCURBITA MAXIMA AND RAPHANUS SATIVUS

IN VITRO QUALITATIVE AND QUANTITATIVE PHYTOCHEMICAL ANALYSIS OF ETHANOLIC AND 50% ETHANOLIC EXTRACTS OF TINOSPORA CORDIFOLIA, MOMORDICA CHARANTIA, CUCURBITA MAXIMA AND RAPHANUS SATIVUS

Amol Gurav*¹, D.B. Mondal ¹ and H. Vijayakumar ²

Division of Medicine, Indian Veterinary Research Institute ¹, Bareilly, Uttar Pradesh, India

Resident Veterinary Services Section, Madras Veterinary College ², Chennai, Tamil Nadu, India

ABSTRACT:Phytochemicals are non-nutritive chemical constituents of plants which occur naturally in it such as Steroid, Terpenoids, Tannins, Carotenoids, Flavonoids, Alkaloids and Glycosides. The aim of the present study was to carryout qualitative and quantitative phytochemical analysis of ethanolic and 50% ethanolic extracts of Tinospora cordifolia (stems), Momordica chirantia (fruit), Cucurbita maxima (fruit) and Raphanus sativus (root). The result of the qualitative phytochemical screening revealed the presence of various secondary metabolites with highest activity in 50% ethanolic extracts of T. cordifolia, C. maxima and ethanolic extract of Tinospora cordifolia. Quantitative phytochemical analysis revealed that the total flavonoids content was found to be varied in different extracts wherein high activity was noted in 50% ethanolic extract of T.cordifolia (9.91 ± 1.46 mg CE/gm)followed by 50 % ethanolic extract of Cucurbita maxima (9.79 ± 0.35 mg CE/gm)and absolute ethanolic extract of T.cordifolia (9.75 ± 0.4 mg CE/gm) among all 8 tested extracts. Total phenol content was significantly high in 50% ethanolic extracts of Tinospora cordifolia (24.70 ± 2.04 mg GAE/gm) followed by 50% ethanolic extract of Cucurbita maxima (18.23 ± 0.03 GAE/gm) and absolute ethanolic extract of Tinospora cordifolia (12.33 ± 0.5mg GAE/gm).

Phytochemical, Tinospora cordifolia, Momordica charantia, Cucurbita maxima and Raphanus sativus.

INTRODUCTION: Phytochemicals are bioactive substances of plants that have been associated in the protection of human and animal health against chronic degenerative diseases. The major groups of phytochemicals that may contribute to the total antioxidant capacity (TAC) of plant foods include polyphenols, carotenoids and the traditional antioxidant vitamins such as vitamin C and vitamin E. Tinospora cordifolia (Hindi name–Guduchi)is a climbing deciduous shrub.

It is found throughout tropical part of India and also found in China, Bangladesh, Myanmar & Srilanka. This plant belongs to the family Menispermaceae ¹. Guduchi is widely used in veterinary folk medicine/ayurvedic system of medicine for its general tonic, antiperiodic, anti-spasmodic, anti-inflammatory, antiarthritic, anti-allergic and anti-diabetic properties ^2-3.Momordica charantia (Karela) commonly known as bitter gourd, bitter melon or balsam pear is an economically important medicinal plant belonging to the family Cucurbitaceae ⁴. The local traditional practitioners use the fruit of Momordiaca Charantia as a medicine for a variety of disorders. Momordica Charantia is a well-known to possess antihyperglycemia, anticholesterol, immuno-suppressive, antiulcerogenic, anti-HIV, anti-ulcer, antiinflammatory, anti-leukemic, anti-microbial, anticholesterol, immunosuppresive, and anti-tumor activities ⁵. Cucurbita Maxima (Pumpkin) belongs to the genus of Cucurbita and the family Cucurbitaceae and it is monoecious. R. sativus niger belongs to family Brassicaceae (order -Brassicales, subspecies-niger and variety-niger). It is a food crop, mostly an ingredient of salads in Asian countries during winter. Aim of the present study was to determine phytochemical compounds qualitatively and quantitatively, by in vitro testing in various vegetable/plant extracts.

MATERIALS AND METHODS:

Plant materials: Following plant materials viz., Tinospora cordifolia (stem), Momordica charantia (fruit), Cucurbita maxima (fruit), Raphanus Sativus (root) were collected in and around Bareilly (U.P) area and air dried and grinded to powder form and were subjected to extraction by absolute ethanolic and 50% ethanolic by columnar soxhlet method at temperature of 40-41^oC with standard protocol. The extracts were dried at 41⁰C temperature.

In vitro qualitative phytochemical tests:

1. Dragendroff’s Test:Extracts were dissolved individually in N/10 Hydrochloric acid and filtered. 1-2 ml filtrates were treated with Dragendroff’s reagent (solution of Potassium Bismuth Iodide). Formation of red precipitate indicated the presence of alkaloids ⁶_.
2. FeCl₃ Test:Extracts were dissolved individually in N/10 Hydrochloric acid and filtered. 1-2 ml filtrates were treated with 5% FeCl₃. Appearance of greenish black ring indicated the presence of phenols ⁶.
3. Foam Test:0.5 gm of extract was shaken with 2 ml of water. If foam produced persisted for ten minutes it indicated the presence of saponins ⁶_.
4. Gelatin Test: To the extract, 1% gelatin solution containing sodium chloride was added. Formation of white precipitate indicated the presence of tannins ⁶.

In vitro quantitative phytochemical tests:

1. Total Flavonoids Content (TFC): The total flavonoids content of the extracts and the potencies were determined according to colorimetric method ⁷. In brief, the sample solution (0.5 ml) was mixed with distilled water (2 ml) andsubsequently with 5% NaNO₂ solution (0.15 ml). After 6 min of incubation, 10% AlCl₃ solution (0.15ml) was added and then allowed to stand for 6 min, followed by additon of 4% NaOH solution (2 ml)to the mixture. Consequently, water was added to the sample to bring the final volume to 5 ml and themixture was thoroughly mixed and allowed to stand for another 15 min. The mixture’s absorbance wasdetermined at 510 nm. The total flavonoid content was expressed in mg of catechin equivalent (CE) per gram ofextract.
2. Total Phenol Content (TPC): The total phenolic content in the extracts and the potencies was measured using Folin-Ciocalteu reagentmethod ⁸. The samples (0.4 mL) (1mg/mL extracts) were transferred into testtubes. To this solution, distilled water (1.0 mL) and Folin-Ciocalteu reagent (1.0 mL) were added, andthe tubes shaken thoroughly. After 1 min, sodium carbonate solution (Na₂CO₃, 1.6 mL, 7.5%) wasadded and the mixture was allowed to stand for 30 min with intermittent shaking. A linear dose responseregression curve was generated using absorbance reading of gallic acid at the wavelength of 765 nm using UV spectrophotometer. The total phenolic compoundsconcentration in the extract and potencies was expressed as milligrams of gallic acid equivalent per gram of dryweight of extract (mg GAE/g).

RESULTS: The presences of different phytochemical compounds in 8 tested plant extracts are shown in Table 1 and Figure 1. Highly positive (++) alkaloids were noticed in 50 % ethanolic extracts of T.cordifolia, C.maxima (50% ethanolic) and ethanolic extract of R.Sativus. Ethanolic and 50% ethanolic extract of M.charantia showed positive (+) alkaloid reaction by Dragendroff’s method.

Highly positive (++) phenols were noticed in ethanolic and 50% ethanolic extracts of T.cordifolia and M.charantia alongwith 50% ethanolic extract of R.Sativus. Ethanolic and 50% ethanolic extracts of Cucurbita maxima showed positive (+) reaction for phenolic compoundswhereas Raphanus sativus (Absolute ethanolic) showed negative (-) reaction for phenols. Highly positive (++) reaction for saponins was recorded in ethanolic and 50% ethanolic extract of T.cordifolia. Positive (+) reaction was noticed only in 50% ethanolic extract of C.maxima whereas ethanolic extract of C.maxima and both the ethanolic and 50% ethanolic extracts of M. charantia and R.sativus revealed negative (-) saponin reaction by foam test. Both extracts of R. sativus (ethanolic and 50% ethanolic) revealed highly positive (++) reaction for tannins. All other extracts showed positive (+) reaction for tannins.

TABLE 1: IN VITRO QUALITATIVE PHYTOCHEMICAL ANALYSIS OF VARIOUS ETHANOLIC (E) AND 50 % ETHANOLIC (A: E) EXTRACTS

(+) positive, (++) highly positive and (-) Negative

FIGURE 1: DIFFERENT QUALITATIVE PHYTOCHEMICAL TESTS FOR TANNINS, ALKALOIDS, SAPONINS & PHENOLS

Total flavonoid contents of the extracts were determined according to colorimetric method and expressed in mg of catechin equivalent per gm (mg CE/gm) of extract, by reference to standard curve (y = 0.008x + 0.016 and R2 = 0.927). The presences of catechin content of extracts revealed highest content in 50% ethanolic extract of Tinospora cordifolia (9.91±1.46 mg CE/gm)followed by 50% ethanolic extract of Cucurbita maxima (9.79±0.35 mg CE/gm) and ethanolic extract of Tinospora cordifolia (9.75±0.4 mg CE/gm) among all the 8 tested extracts (Table 2).

Total phenol content, as determined by the Folin-Ciocalteu method ,is reported as gallic acid equivalents (mg GAE/g) by reference to standard curve (y = 0.045x - 0.006 and R² = 0.983; where y is absorbance, X is concentration and R² is the coefficient of determination) as shown in Fig.3. Total phenolic content was significantly (p<0.05) higher in 50% ethanolic extracts of Tinospora cordifolia (24.70 ± 2.04 mg GAE/gm) followed by 50% ethanolic extract of Cucurbita maxima (18.23 ± 0.03  GAE/gm) and absolute ethanolic extract of Tinospora cordifolia (12.33  ± 0 .5mg GAE/gm) (Table 2).

TABLE 2: IN VITRO QUANTITATIVE PHYTOCHEMICAL ANALYSIS OF DIFFERENT VEGETABLE/PLANT EXTRACTS (MEAN ±SE)

Values bearing different superscripts in the same column differ significantly (P≤0.05)

DISCUSSION: Previous study revealed the presence of alkaloids, phenols and saponins in absolute ethanolic, methanolic and acetone extracts of Tinospora cordifolia stems. However same author recorded negative (-) Mayer’s test for hydroethanolic and aqueous extract of Tinospora cordifolia (stems) which is concurrent with present study ⁹. Positive (+) reactions for Dragendroff’s and Lead acetate test were recorded in hydroethanolic and aqueous extract of Tinospora cordifolia (stems) in earlier study ¹⁰. In contrary to our reports, one study reported absence of saponin glycosides in T. cordifolia extracts ¹⁰.

Fruit extract of Momordica charantia revealed presence of alkaloids, steroids, phenolic compounds, flavonoids tannins, anthraquinones and amino acids ¹¹. One of previous in vitro phytochemical analysis revealed the presence of tannins, saponins, flavonoids, anthraquinones, carbohydrates, steroids, phytosterol, alkaloids, amino acids, terpenoids, cardiac glycosides and chalcones in R.sativus ¹². In present study, phenolic content was negative in 50% ethanolic extract of Raphanus sativus which is in concurrence with earlier report who also revealed absence of phenolic compounds in Raphanus sativus ¹². Negative (-) saponin reactionin aqueous fruit extracts of R. sativus and M. charantia revealed in earlier study supports present findings ¹³.

One study reported the presence of chemical constituents like alkaloids, saponins, tannins, flavonoids, terpenoids, glycosides, steroids and phenolic compounds in Pumpkin (Cucurbita maxima) fruits ¹⁴. Several reports confirmed the presence of phenolic compounds in fruits of M.charantia and C. maxima ^{11, 14}.

Positive (+) ferric chloride test for C. maxima indicated presence of phenolic compounds in earlier study is in accordance with the present findings ¹⁵. Earlier studies recorded negative (-) foam reaction in C. maxima and M. charantia extracts ^{11, 15}.  R. sativus ^{12, 13}, M.chirantia ^{11, 12}, T. cordifolia ^{9, 10} and C. maxima ¹⁴ are reported for presence of tannins by several authors from time by time are in accordance with present findings.

Total phenol and Flavonoids contents (TPC/TFC)were high in 50% ethanolic extracts of Tinospora cordifolia and Cucurbita maxima. Earlier study revealed 12.8mg/gm of total phenolic contents in T. cordiflolia extract however total flavonoid content recorded was 6mg/gm of T. cordifolia extract ⁹. Total phenol content (TPC) of T. cordifolia grown on Azadirachta indica was found to be 21.5 mg/gm of dry weight of extract ¹⁶. Our findings are in agreement with above authors. It is known that total phenol content is responsible for the free radical scavenging activity in many plants ¹⁷.

Total polyphenolic, flavonoid and flavonol content of Cucurbita maxima found to be 12mg /gm (gallic acid equivalent),3.8 mg/gm and 0.8 mg/gm (rutin equivalent) respectively. Quantification of phenolics in Cucurbita maxima by Folin-Ciocalteau method showed the presence of good amount of total phenolics (12mg/gm calculated as per gallic acid), total flavonoid and flavonol content found to be 3.8 mg/gm and 0.8 mg/gm (rutin equivalent) respectively ¹⁸.

CONCLUSION: In vitro qualitative and quantitative phytochemical analysis of different vegetable/herb extracts revealed highest activity in 50% ethanolic extract of T. cordifolia > C. maxima (A:E)>T. cordifolia (E) among 8 different extracts.

ACKNOWLEDGEMENT: The authors are thankful to Director and Joint Director (Academic), IVRI for providing necessary facilities and ICAR for providing fellowship.

REFERENCES:

1. Spandana U, Shaik LA, Nirmala T: A Review on Tinospora cordifolia. International Journal of Current Pharmaceutical Review and Research 2013; 4(2):61-68.
2. Kirtikar KR and Basu BD: Indian Medicinal Plants Vol.1. Lalit Mohan Basu, Allahabad India 1984; 677-680.
3. Zhao TF, Wang X, Rimando AM and Che C: Folkloric medicinal plants: Tinospora sagittata var. cravaniana and Mahonia bealei: Planta Med 1991; 57:505.
4. Mada SB, Garba A, Mohammed HA, Muhammad A, Olagunju A and Muhammad AB: Antimicrobial activity and phytochemical screening of aqueous and ethanol extracts of Momordica charantia L. leaves. Journal of Medicinal Plants Research 2013; 7:579-586.
5. Scartezzini P and Speroni E: Review on some plants of Indian traditional medicine with antioxidant activity. Journal of Ethnopharmacology 2000; 71: 23-43.
6. Tiwari P, Kumar B, Kaur M, Kaur G and Kaur H: Phytochemical screening and Extraction: A Review. Internationale Pharmaceutica Sciencia 2011; 1 (1):98-106.
7. Nabavi SM, Ebrahimzadeh MA, Nabavi SF and Jafari M: Free Radical Scavenging Activity And Antioxidant Capacity Of Eryngium Caucasicum Trautv And Froripia Subpinnata. Pharmalacologyonline 2008; 3: 19-25.
8. Biglari F, Karkhi AFM and Easa AM: Antioxidant activity and phenolic content of various date palms (Phoenix dactylifera) fruits from Iran. Food Chemistry 2008; 107:1636-41.
9. Yadav RNS and Agarwala M: Phytochemical analysis of some medicinal plants. Journal of Phytology 2011; 3(12): 10-14.
10. Tanwar S, Jain J, Verma S and Solanki D: Standardization and phytochemical evaluation of Tinospora cordifolia (willd.) Miers. (menispermaceae). International journal of pharmacy and pharmaceutical sciences 2012;4(1):219-233.
11. Britto J and Gracelin HS: Phytochemical analysis and antibacterial activity of Momordica charantia descourt., a know n medicinal plant. Journal of basic and applied biology 2012; 5 :( 1/ 2) 307-311.
12. Janjua S, Shahid M and Abbas F: Phytochemical analysis and in vitro antibacterial activity of root peel extract of Raphanus sativus L. varniger. Advancement in Medicinal Plant Research 2013; 1(1):1-7.
13. Raghu KL, Ramesh CK, Srinivasan TR and Jamuna KS: DPPH scavenging and reducing power properties in common vegetables. Research Journal of Pharmaceutical, Biological and Chemical Sciences 2010; 1(4): 399-405.
14. Singh K : Phytochemical determination and antibacterial activity of trichosanthes dioica roxb (patal), Cucurbita maxima (pumpkin) and Abelmoschus esculentus moench (okra) plant seeds. Thesis, M.Sc (Life Science) 2013; National institute of technology rourkela-769 008, Odisha, INDIA.
15. Bajpai R, Jain N and Pathak AK: Standardization of Ethanolic Extract of Cucurbita Maxima Seed. Journal of Applied Pharmaceutical Science 2012; 2(08): 92-95.
16. Bhalerao BM, Kasote DM, Nagarkar BE, Jagtap SD, Vishwakarma KS, Pawar PK and Maheshwari VL:Comparative analysis of radical scavenging and immunomodulatory activities of Tinospora cordifolia growing with different supporting trees. Acta Biologica Szegediensis 2012; 56(1):  65-71.
17. Adedpo AA, Jlmoh FO, Folayan AJ and Masika PJ:Antioxidant activities and phenolics contents of the metanolic extracts of stem of Acokanthera oppositifolia and Adenia gummifera. BMC Complement Alternative Medicine 2008; 8:54-60.
18. Attarde D, Kadu S, Chaudhari B, Kale S and Bhamber R: In vitro antioxidant activity of pericarp of Cucurbita maxima Duch. ex Lam. International Journal of Pharmaceutical Tech Res. 2010; 2(2):  1533-8.It is found throughout tropical part of India and also found in China, Bangladesh, Myanmar & Srilanka. This plant belongs to the family Menispermaceae ¹. Guduchi is widely used in veterinary folk medicine/ayurvedic system of medicine for its general tonic, antiperiodic, anti-spasmodic, anti-inflammatory, antiarthritic, anti-allergic and anti-diabetic properties ^2-3.Momordica charantia (Karela) commonly known as bitter gourd, bitter melon or balsam pear is an economically important medicinal plant belonging to the family Cucurbitaceae ⁴. The local traditional practitioners use the fruit of Momordiaca Charantia as a medicine for a variety of disorders. Momordica Charantia is a well-known to possess antihyperglycemia, anticholesterol, immuno-suppressive, antiulcerogenic, anti-HIV, anti-ulcer, antiinflammatory, anti-leukemic, anti-microbial, anticholesterol, immunosuppresive, and anti-tumor activities ⁵. Cucurbita Maxima (Pumpkin) belongs to the genus of Cucurbita and the family Cucurbitaceae and it is monoecious. R. sativus niger belongs to family Brassicaceae (order -Brassicales, subspecies-niger and variety-niger). It is a food crop, mostly an ingredient of salads in Asian countries during winter. Aim of the present study was to determine phytochemical compounds qualitatively and quantitatively, by in vitro testing in various vegetable/plant extracts.MATERIALS AND METHODS:Plant materials: Following plant materials viz., Tinospora cordifolia (stem), Momordica charantia (fruit), Cucurbita maxima (fruit), Raphanus Sativus (root) were collected in and around Bareilly (U.P) area and air dried and grinded to powder form and were subjected to extraction by absolute ethanolic and 50% ethanolic by columnar soxhlet method at temperature of 40-41^oC with standard protocol. The extracts were dried at 41⁰C temperature.In vitro qualitative phytochemical tests:
    1. Dragendroff’s Test:Extracts were dissolved individually in N/10 Hydrochloric acid and filtered. 1-2 ml filtrates were treated with Dragendroff’s reagent (solution of Potassium Bismuth Iodide). Formation of red precipitate indicated the presence of alkaloids ⁶_.
    2. FeCl₃ Test:Extracts were dissolved individually in N/10 Hydrochloric acid and filtered. 1-2 ml filtrates were treated with 5% FeCl₃. Appearance of greenish black ring indicated the presence of phenols ⁶.
    3. Foam Test:0.5 gm of extract was shaken with 2 ml of water. If foam produced persisted for ten minutes it indicated the presence of saponins ⁶_.
    4. Gelatin Test: To the extract, 1% gelatin solution containing sodium chloride was added. Formation of white precipitate indicated the presence of tannins ⁶.

    In vitro quantitative phytochemical tests:

    1. Total Flavonoids Content (TFC): The total flavonoids content of the extracts and the potencies were determined according to colorimetric method ⁷. In brief, the sample solution (0.5 ml) was mixed with distilled water (2 ml) andsubsequently with 5% NaNO₂ solution (0.15 ml). After 6 min of incubation, 10% AlCl₃ solution (0.15ml) was added and then allowed to stand for 6 min, followed by additon of 4% NaOH solution (2 ml)to the mixture. Consequently, water was added to the sample to bring the final volume to 5 ml and themixture was thoroughly mixed and allowed to stand for another 15 min. The mixture’s absorbance wasdetermined at 510 nm. The total flavonoid content was expressed in mg of catechin equivalent (CE) per gram ofextract.
    2. Total Phenol Content (TPC): The total phenolic content in the extracts and the potencies was measured using Folin-Ciocalteu reagentmethod ⁸. The samples (0.4 mL) (1mg/mL extracts) were transferred into testtubes. To this solution, distilled water (1.0 mL) and Folin-Ciocalteu reagent (1.0 mL) were added, andthe tubes shaken thoroughly. After 1 min, sodium carbonate solution (Na₂CO₃, 1.6 mL, 7.5%) wasadded and the mixture was allowed to stand for 30 min with intermittent shaking. A linear dose responseregression curve was generated using absorbance reading of gallic acid at the wavelength of 765 nm using UV spectrophotometer. The total phenolic compoundsconcentration in the extract and potencies was expressed as milligrams of gallic acid equivalent per gram of dryweight of extract (mg GAE/g).

    RESULTS: The presences of different phytochemical compounds in 8 tested plant extracts are shown in Table 1 and Figure 1. Highly positive (++) alkaloids were noticed in 50 % ethanolic extracts of T.cordifolia, C.maxima (50% ethanolic) and ethanolic extract of R.Sativus. Ethanolic and 50% ethanolic extract of M.charantia showed positive (+) alkaloid reaction by Dragendroff’s method.

    Highly positive (++) phenols were noticed in ethanolic and 50% ethanolic extracts of T.cordifolia and M.charantia alongwith 50% ethanolic extract of R.Sativus. Ethanolic and 50% ethanolic extracts of Cucurbita maxima showed positive (+) reaction for phenolic compoundswhereas Raphanus sativus (Absolute ethanolic) showed negative (-) reaction for phenols. Highly positive (++) reaction for saponins was recorded in ethanolic and 50% ethanolic extract of T.cordifolia. Positive (+) reaction was noticed only in 50% ethanolic extract of C.maxima whereas ethanolic extract of C.maxima and both the ethanolic and 50% ethanolic extracts of M. charantia and R.sativus revealed negative (-) saponin reaction by foam test. Both extracts of R. sativus (ethanolic and 50% ethanolic) revealed highly positive (++) reaction for tannins. All other extracts showed positive (+) reaction for tannins.

    
    TABLE 1: IN VITRO QUALITATIVE PHYTOCHEMICAL ANALYSIS OF VARIOUS ETHANOLIC (E) AND 50 % ETHANOLIC (A: E) EXTRACTS

    Extract (s) /Test(s)	Alkaloids Dragendroff’s Test	Phenols FeCl3 Test	Saponins Foam Test	Tannins Gelatin Test
    T. cordifolia (E)	_	++	++	+
    T. cordifolia (A:E)	++	++	++	+
    M. chirantia (E)	+	++	_	+
    M. chirantia (A:E)	+	++	_	+
    C. maxima (E)	_	+	_	+
    C. maxima (A:E)	++	+	+	+
    R. Sativus (E)	++	_	_	++
    R. sativus (A:E)	_	++	_	++

    (+) positive, (++) highly positive and (-) Negative

    FIGURE 1: DIFFERENT QUALITATIVE PHYTOCHEMICAL TESTS FOR TANNINS, ALKALOIDS, SAPONINS & PHENOLS
    Total flavonoid contents of the extracts were determined according to colorimetric method and expressed in mg of catechin equivalent per gm (mg CE/gm) of extract, by reference to standard curve (y = 0.008x + 0.016 and R2 = 0.927). The presences of catechin content of extracts revealed highest content in 50% ethanolic extract of Tinospora cordifolia (9.91±1.46 mg CE/gm)followed by 50% ethanolic extract of Cucurbita maxima (9.79±0.35 mg CE/gm) and ethanolic extract of Tinospora cordifolia (9.75±0.4 mg CE/gm) among all the 8 tested extracts (Table 2).

    Total phenol content, as determined by the Folin-Ciocalteu method ,is reported as gallic acid equivalents (mg GAE/g) by reference to standard curve (y = 0.045x - 0.006 and R² = 0.983; where y is absorbance, X is concentration and R² is the coefficient of determination) as shown in Fig.3. Total phenolic content was significantly (p<0.05) higher in 50% ethanolic extracts of Tinospora cordifolia (24.70 ± 2.04 mg GAE/gm) followed by 50% ethanolic extract of Cucurbita maxima (18.23 ± 0.03  GAE/gm) and absolute ethanolic extract of Tinospora cordifolia (12.33  ± 0 .5mg GAE/gm) (Table 2).

    
    TABLE 2: IN VITRO QUANTITATIVE PHYTOCHEMICAL ANALYSIS OF DIFFERENT VEGETABLE/PLANT EXTRACTS (MEAN ±SE)

    Plant Extracts/ Test (s)	Total Phenols (mg GAE/gm)	Total Flavonoids (mg CE/gm)
    M. chirantia (A:E)	11.54 ± 0.007c	4.68 ± 0.10ab
    C. maxima (E)	7.34±.04b	7.49 ± 0.20bc
    C. maxima (A:E)	18.23 ± 0.03d	9.79 ± 0.35c
    R. sativus (E)	4.22 ± 0.06ab	2.49 ± 0.32a
    R. sativus (A:E)	3.28 ±0.29a	1.82 ± 0.05ac
    M. chirantia (E)	4.53 ± 0.36ab	7.26 ± 0.4b
    T. cordifolia (E)	12.33  ±  0 .5c	9.75  ±  0.4c
    T. cordifolia (A:E)	24.70  ± 2.04e	9.91 ± 1.46c

    Values bearing different superscripts in the same column differ significantly (P≤0.05)

    
    DISCUSSION: Previous study revealed the presence of alkaloids, phenols and saponins in absolute ethanolic, methanolic and acetone extracts of Tinospora cordifolia stems. However same author recorded negative (-) Mayer’s test for hydroethanolic and aqueous extract of Tinospora cordifolia (stems) which is concurrent with present study ⁹. Positive (+) reactions for Dragendroff’s and Lead acetate test were recorded in hydroethanolic and aqueous extract of Tinospora cordifolia (stems) in earlier study ¹⁰. In contrary to our reports, one study reported absence of saponin glycosides in T. cordifolia extracts ¹⁰.

    Fruit extract of Momordica charantia revealed presence of alkaloids, steroids, phenolic compounds, flavonoids tannins, anthraquinones and amino acids ¹¹. One of previous in vitro phytochemical analysis revealed the presence of tannins, saponins, flavonoids, anthraquinones, carbohydrates, steroids, phytosterol, alkaloids, amino acids, terpenoids, cardiac glycosides and chalcones in R.sativus ¹². In present study, phenolic content was negative in 50% ethanolic extract of Raphanus sativus which is in concurrence with earlier report who also revealed absence of phenolic compounds in Raphanus sativus ¹². Negative (-) saponin reactionin aqueous fruit extracts of R. sativus and M. charantia revealed in earlier study supports present findings ¹³.

    One study reported the presence of chemical constituents like alkaloids, saponins, tannins, flavonoids, terpenoids, glycosides, steroids and phenolic compounds in Pumpkin (Cucurbita maxima) fruits ¹⁴. Several reports confirmed the presence of phenolic compounds in fruits of M.charantia and C. maxima ^{11, 14}.

    Positive (+) ferric chloride test for C. maxima indicated presence of phenolic compounds in earlier study is in accordance with the present findings ¹⁵. Earlier studies recorded negative (-) foam reaction in C. maxima and M. charantia extracts ^{11, 15}.  R. sativus ^{12, 13}, M.chirantia ^{11, 12}, T. cordifolia ^{9, 10} and C. maxima ¹⁴ are reported for presence of tannins by several authors from time by time are in accordance with present findings.

    Total phenol and Flavonoids contents (TPC/TFC)were high in 50% ethanolic extracts of Tinospora cordifolia and Cucurbita maxima. Earlier study revealed 12.8mg/gm of total phenolic contents in T. cordiflolia extract however total flavonoid content recorded was 6mg/gm of T. cordifolia extract ⁹. Total phenol content (TPC) of T. cordifolia grown on Azadirachta indica was found to be 21.5 mg/gm of dry weight of extract ¹⁶. Our findings are in agreement with above authors. It is known that total phenol content is responsible for the free radical scavenging activity in many plants ¹⁷.

     

    Total polyphenolic, flavonoid and flavonol content of Cucurbita maxima found to be 12mg /gm (gallic acid equivalent),3.8 mg/gm and 0.8 mg/gm (rutin equivalent) respectively. Quantification of phenolics in Cucurbita maxima by Folin-Ciocalteau method showed the presence of good amount of total phenolics (12mg/gm calculated as per gallic acid), total flavonoid and flavonol content found to be 3.8 mg/gm and 0.8 mg/gm (rutin equivalent) respectively ¹⁸.

     

    CONCLUSION: In vitro qualitative and quantitative phytochemical analysis of different vegetable/herb extracts revealed highest activity in 50% ethanolic extract of T. cordifolia > C. maxima (A:E)>T. cordifolia (E) among 8 different extracts.

     

    ACKNOWLEDGEMENT: The authors are thankful to Director and Joint Director (Academic), IVRI for providing necessary facilities and ICAR for providing fellowship.

    REFERENCES:

    1. Spandana U, Shaik LA, Nirmala T: A Review on Tinospora cordifolia. International Journal of Current Pharmaceutical Review and Research 2013; 4(2):61-68.
    2. Kirtikar KR and Basu BD: Indian Medicinal Plants Vol.1. Lalit Mohan Basu, Allahabad India 1984; 677-680.
    3. Zhao TF, Wang X, Rimando AM and Che C: Folkloric medicinal plants: Tinospora sagittata var. cravaniana and Mahonia bealei: Planta Med 1991; 57:505.
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        How to cite this article:

        Gurav A, Mondal DB and Vijayakumar H: In vitro qualitative and quantitative phytochemical analysis of ethanolic and 50% ethanolic extracts of tinospora cordifolia, momordica charantia, Cucurbita maxima and Raphanus sativus.Int J Pharm Sci Res 2014; 5(5): 1937-41.doi: 10.13040/IJPSR.0975-8232.5 (5).1937-41.

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