e-ISSN: 2582-5208 International Research Journal of Modernization in Engineering Technology and Science ( Peer-Reviewed, Open Access, Fully Refereed International Journal ) Volume:03/Issue:09/September-2021 Impact Factor- 6.752 www.irjmets.com QUALITATIVE, QUANTITATIVE ANALYSIS AND CYTOTOXIC ACTIVITY OF LEAVES EXTRACT OF CORDIA DICHOTOMA Mr. Lingraj V. Sargar*1, Miss. Priyanka V. Bagade*2, Mr. Sahebrao H. Shembade*3, Ashapak M. Tamboli*4, Manoj A. Patil*5 *1Student, Pharmaceutics, Bharti Vidyapeeth College Of Pharmacy Kolhapur,Kolhapur, Maharashtra, India. *2Student, *3Student, Pharmaceutics, Rajarambapu College Of Pharmacy Kasegaon,Maharashtra, India. Pharmaceutics, Sahyadri College Of Pharmacy Methwade, Sangola, Maharashtra, India. *4Professor And Head Department Of Pharmaceutical Chemistry, Sahyadri College Of Pharmacy Methwade, Sangola, Maharashtra, India. *5Professor And Head Department Of Pharmaceutics, Sahyadri College Of Pharmacy Methwade, Sangola, Maharashtra, India. ABSTRACT Because there is a scarcity of scientific data on Cordia, a qualitative and quantitative analysis of the leaves is required was performed by using the method given in WHO (1998) and Trease and Evans (2002). The plant of Cordia dichotoma was collected from Sahyadri College of Pharmacy Methwade, Sangola, Solapur. Identification of Cordia dichotoma was done by standard procedures. In phytochemical screening four different extract such as ethyl acetate, chloroform, ethanol and aqueous extract of leaves contain tannin, flavonoids Alkaloids were found in a 1 percent hydrochloric acid extract of leaves, although steroids were absent. Because of the genus Cordia's ethanol pharmacological and chemotaxonomic relevance, we looked at the chemical and secondary contents of one of its species. In quantitative determination total alkaloid and flavonoid content were estimated from the crude leaves powder of Cordia dichotoma (G. forst). The cytotoxicity of Cordia dichotoma leaves extract was determined using a brine shrimp lethality test. Keywords: Phytochemical, Cytotoxic, Cordia, G. Forst. I. INTRODUCTION Cordia is a genus of flowering plants, belongs to the family Boraginaceae [1]. Cordia G. forst; is a perennial tree, native to India's tropical and subtropical regions. The leaf has been used for wrap cheroot. The chemical screening of both leaves and fruits reveals presence of alkaloids, flavonoids, saponins, sterols [2]. On the basis of literature survey number of medicinal uses reported by different cordia species [3, 4, 5]. It also thrives in Maharashtra's damp monsoon jungle. It does not grow in clumps, but rather grows alone in wet, dark ravines and valleys [6]. The fruits of Cordia dichotoma are used as cooling, astringent, emollient, anthelmintic, expectorant, diuretic agent and purgative. [7]. It has been determined that all sections of Cordia dichotoma have been used in various treatments. The ripe fruit, as well as the bark, are employed in tonic preparations. The preparation of leaves is beneficial in the treatment of infections and headaches. The plants contain phenolic compounds has important properties like antithrombotic, cardio protective and vasodilatory effect [8, 9]. Cordia G. forst. are used as anti-ulcer [10, 11] anti-inflammatory and analgesic [12, 13, 14, 15] anticancer [16] antimicrobial [17] hepatoprotective and diuretic purposes [18, 19]. Cancer is the one of the leading causes of death [20]. Chemotherapy is an important option for the treatment of cancer apart from surgical operation and irradiation. Medicinal plants are the one of major source of chemotherapy drugs in modern as well as traditional medicine [21]. Currently, the MTT test is frequently utilized in cell viability and proliferation research. [22]. More than 50% of breast cancer cases occurred in developing countries. The development of the disease is influenced by both lifestyle and hormonal risk factors. [23]. Several studies on the chemical composition of Cordia species related to leaves and fruits were found [24]. As a result, the purpose of this study is to look into the qualitative and quantitative analysis of Cordia G. forst. www.irjmets.com @International Research Journal of Modernization in Engineering, Technology and Science [900] e-ISSN: 2582-5208 International Research Journal of Modernization in Engineering Technology and Science ( Peer-Reviewed, Open Access, Fully Refereed International Journal ) Volume:03/Issue:09/September-2021 Impact Factor- 6.752 www.irjmets.com II. METHODOLOGY a) Sample collection and preparation: Tablets The fresh leaves of cordial dichotoma were collected from Sahyadri College of Pharmacy, Methwade, Sangola during August 2020. The plant was identified by the Dr. Tembhurne R.R. Department of Botany, Sangola College Sangola with the help of flora of Solapur District Maharashtra India and a voucher The specimen was taken to the herbarium, Department of Botany, Sangola College Sangola. The leaves were washed, cut into little pieces, and air dried for several days at room temperature. After being completely dried they were pulverized by grinder machine to get powder and stored in a air tight container to prevent it from moisture and avoid contamination. b) Preparation of Extracts: 50 gm of powdered Cordia dichotoma leaves were extracted with 250ml of ethyl acetate, ethanol and chloroform solvent as a separately and maintain the temperature between 65 and 70℃for 24 hours by using a Soxhlet extractor. To obtain viscous semi-solid masses, the solvent was evaporated using a rotary evaporator. This semi-dry crude extract was used to phytochemical screening of the plant. For aqueous preparation 250gm of coarse powder is macerated with 500ml of water for 24 hours. Add chloroform to avoid bacterial growth. Then concentrate the extract for further use. III. MODELING AND ANALYSIS Phytochemical Screening: Qualitative Analysis of Phytochemicals: In present study five secondary metabolites was analyzed qualitatively and quantitatively. The extract of leaves was analyzed for the occurrence of numerous phytoconstituents like alkaloid, glycosides, flavonoid, saponin and sterol. Test for Alkaloids: a) Wagner’s Test: 3-4 drops of Wagner’s reagent was added to extract. Alkaloid presence was confirmed after the development of yellow or brown precipitate [25, 26, 27]. b) Mayer’s Test: To extract add 3-4 drops of Mayer’s reagent. Alkaloid presence was confirmed after the formation of white or pale-yellow precipitate. Test of Glycosides: a) Legal’s Test: To the extract add 1ml of pyridine and add mixture of sodium nitroprusside and sodium hydroxide. The presence of glycosides is indicated by the formation of a pink to red colour. b) Keller-Killiani test: To 2ml of extract, add glacial acetic acid, one drop 5% Fecl3 and Conc. H2SO4. Reddish brown colour appears at the junction of the two liquid layers and upper layer appears bluish green. Test for Flavonoid: a) Shinoda’s Test: To 5ml of ethanol add extract and dissolved. To these ten drops of dilute hydrochloric acid was added, it was followed by addition of small piece of magnesium. The presence of flavonoid was confirmed after the development of brown reddish or pink colour [25, 26, 27]. b) Alkaline Test: A few drops of sodium hydroxide solution were added to the extract. Development of intense yellow colour, When dilute acid is added, it turns colourless, indicating the presence of flavonoids. Test of Saponin a) Foam test: Shake the drug extract vigorously with water. Persistent foam observed. Test for Sterols: a) Salkowski test: To 2ml extract, add 2ml chloroform and 2ml conc. H2SO4 and shake well. The chloroform layer fluoresces red, while the acid layer fluoresces greenish yellow. b) Libermann-Burchard test: Mix 2ml extract with chloroform. From the side of the tube, add 1-2ml acetic anhydride and 2 drops of conc. H2SO4. First the development of red, then blue and finally green colour appears. Quantitative Analysis: A) Alkaloid determination: The quantification of alkaloid was carried out according to methodology used by Harbome. 100ml of ten www.irjmets.com @International Research Journal of Modernization in Engineering, Technology and Science [901] e-ISSN: 2582-5208 International Research Journal of Modernization in Engineering Technology and Science ( Peer-Reviewed, Open Access, Fully Refereed International Journal ) Volume:03/Issue:09/September-2021 Impact Factor- 6.752 www.irjmets.com percent (10%) acetic acid in ethanol was added to leaves powder sample (1.25gm) in a 250ml beaker and allowable to stand for four hours. The extract was concentrated in a water bath to obtain one quarter of its original volume, and then 10-15 drops of concentrated Ammonium hydroxide were added to the extract drop by drop until the precipitate was obtained immediately after filtering. After 3 hours the mixture is sediments, the upper layer was discarded and precipitate was washed with 10ml of 0.1M Whatman filter paper no-42 was used to filter the ammonium hydroxide. The extract was dried in oven and % of alkaloid is represented mathematically by, Weight of alkaloids Percent alkaloids = --------------------------- X 100 Weight of sample B) Flavonoid determination: Determination of flavonoid was done by the method reported by the Ejikeme et al. and Kocipai and Boham. 50ml of 80% aqueous methanol was added to 2.5gm of leaves powder in 250ml beaker, covered and let to stand at room temperature for 24 hours. After discarding the supernatant, the residue was re-extracted with the similar volume of ethanol (3 times). Whatman filter paper number 42 was used to filter the whole solution of the leaves sample. Then leaves sample filtrate was transferred to the crucible and evaporated to aridness over water bath. Then content in the crucible was chilled in desiccators and weight. % of flavonoid was calculated by, Weight of flavonoid Percent flavonoid = ---------------------------- X 100 Weight of Sample BRINE SHRIMP LETHALITY BIOASSAY: This study was studied to investigate cytotoxic properties of Cordia dichotoma in ethanol extract. The cytotoxicity has been assessed by using brine shrimp lethality bioassay. Preparation of reagents: Preparation of sea water: 38 gm of sea salt was weighed, dissolved in one liter of distilled water and filtered off to get clear solution. Hatching of Brine Shrimp: The test organism was brine shrimp eggs obtained from pet stores. The little tank was filled with sea water, and shrimp eggs were placed on one side of the tank, which was then covered. The shrimp were allowed to hatch and mature as nauplii for two days. Throughout the hatching period, a constant oxygen supply was maintained. The hatched shrimps are attached to the light and so nauplii was taken from the fish tank by a pipette and diluted in fresh clear sea water to increase visibility and nauplii were taken carefully by micropipette. Preparation of Standard Solution: 5- Fluorouracil is used as standard. Stock solution of 5- Fluorouracil in ethanol was prepared of 2500µg/ml. From this stock solution 5 different concentrations of 50, 125, 250, 500 and 1250µg/ml were prepared. Preparation of Test Solution: Stock solution of the various leaves extract of 2500µg/ml was prepared by using ethanol as a solvent. From this stock solution 5 different concentrations of 50, 125, 250, 500 and 1250µg/ml were prepared. Procedures: Preparation of test solution absolute ethanol extract: To make a sample of the extract, dissolve 25mg of extract in 20l of DMSO and dilute to 10ml with distilled water to make a stock solution of 2500g/ml. From this stock solution was serial diluted to 50, 125, 250, 500 and 1250µg/ml with sea water. Then 0.5ml solution of plant extract was added to 4.5ml of sea water containing 10 nauplii. Statistical Analysis: The data were analyzed statistically by ANOVA followed by student ‘t’ test with Graph Pad Prism Data Editor for Windows. www.irjmets.com @International Research Journal of Modernization in Engineering, Technology and Science [902] e-ISSN: 2582-5208 International Research Journal of Modernization in Engineering Technology and Science ( Peer-Reviewed, Open Access, Fully Refereed International Journal ) Volume:03/Issue:09/September-2021 Impact Factor- 6.752 www.irjmets.com IV. RESULTS AND DISCUSSION Qualitative Analysis: The result of qualitative phytochemical test of Cordia dichotoma leaves of different extract are given in table1. Quantitative Analysis: The result of quantitative analysis of Cordia dichotoma leaves of powder are shown in table 2. Brine Shrimp Lethality Bioassay: In brine shrimp lethality bioassay using brine shrimp nauplii, the ethanol extract of C. dichotoma leaves showed positive result in comparison with the control that is positive 5- Fluorouracil & that’s why it can be assumed that extract is pharmacologically active. By plotting different concentration verses % of mortality for all test samples showed a linear correlation. The median lethal concentration IC50, the concentration at which 50% mortality of brine shrimp nauplii was calculated, may be seen in the graph. The crude ethanol extract of C. dichotoma showed significant cytotoxic activity against brine shrimp nauplii and IC50 value was 151.13µg/ml (table 3 and figure 1). As 5- Fluorouracil was used & as a negative control DMSO was used to validate the test method. Table 1. Phytochemical test of leaves extract of Cordia dichotoma SN. Chemical constituents Chemical test Ethanol Ethyl acetate Chloroform Aqueous 1 Alkaloids Mayer’s test + - + + + + + + 2 Glycosides Hager’s test Keller-killiani test + + - - + - + + 3 Flavonoids Legal’s test Shinoda’s test + + + - Alkaline test - + + + 4 Saponin Foam test - + - + Salkowski test + + + + Liebermann Burchard test + - + + 5 Sterol (+ve sign is denoted as test is present and –ve sign is denoted as test is absent) Table 2. Result of Quantitative assay of Cordia dichotoma leaves of powder SN. Name of Phytochemical constituents % Of Phytochemical present 1 Alkaloid 1.52 % 2 Flavonoid 4.64 % Table 3. Cytotoxic assay of ethanolic extract Cordia dichotoma No. of nauplii dead T1 T2 T3 No. of nauplii live 10 4 4 2 20 33.33 2.0969 10 5 5 4 16 46.66 250 2.3979 10 6 7 6 11 63.33 151.13 4 500 2.6989 10 8 8 7 7 76.66 µg/ml 5 1250 3.0969 10 7 10 11 2 93.33 Concentration µg/ml Log C 1 50 1.6989 2 125 3 SN. www.irjmets.com Total nauplii % Mortality IC 50 value @International Research Journal of Modernization in Engineering, Technology and Science [903] e-ISSN: 2582-5208 International Research Journal of Modernization in Engineering Technology and Science ( Peer-Reviewed, Open Access, Fully Refereed International Journal ) Volume:03/Issue:09/September-2021 Impact Factor- 6.752 www.irjmets.com Cytotoxic y = 0.0444x + 43.354 R² = 0.828 120 100 80 % Mortality 60 % mortality 40 Linear (% mortality ) 20 0 0 500 1000 Concentration 1500 Figure 1. Linearity curve of cytotoxic assay Table 4. BSL assay of different extract of cordia dichotoma leaves SN. Plant extracts IC 50 values (µg/ml) 1 Standard (5- Fluorouracil) 70µg/ml 2 Ethanol extract 151.13µg/ml 3 Ethyl acetate extract 237.82µg/ml 4 Chloroform extract 242.40µg/ml 5 Aqueous extract 460µg/ml Brine shrimp lethality assay 500 IC 50 µg/ml 400 300 200 100 s eo u rm q u fo A lo h C yl E th ro et at o ac th an ar E d n S ta e l d 0 Extracts Fig 2. IC 50 values of different extract of cordia dichotom leaves V. CONCLUSION In this investigation, the preliminary phytochemical test, quantitative determination of phytochemical constituents and cytotoxic activity of cordial dichotoma (G. forst) has been studied. In Qualitative determination various qualitative phytochemical test are performed by using the solvents chloroform, ethyl acetate and aqueous solvents to detect the optional metabolites such as alkaloids, flavonoids, glycosides, steroids and saponin in the leaves of concentrate of cordial dichotoma (G. forst). 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