Physico and phytochemical standardization of nutritionally rich mulberry fruits

(Morus indica Linn)

Akhlaq Mustafa¹*, Anas Iqbal Alvi², Mahesh Chandera³, Ghazala Javed⁴, Asim Ali Khan⁵

1Research Officer (Chemistry) Drug Standardization Research Unit,

Central Council for Research in Unani Medicine, New Delhi

²Research Assistant (Chemistry) Drug Standardization Research Unit,

Central Council for Research in Unani Medicine, New Delhi

³Laboratory Technician, Drug Standardization Research Unit,

Central Council for Research in Unani Medicine, New Delhi

⁴Research Officer (Unani), Incharge, Drug Standardization Research Unit,

Central Council for Research in Unani Medicine, New Delhi

⁵Director General, Central Council for Research in Unani Medicine, 61- 65,

Institutional Area, Janakpuri, New Delhi

*Corresponding Author E-mail: amh786786@gmail.com

ABSTRACT:

Due to their sweet flavor, impressive nutritional value, and various health benefits, mulberries are gaining popularity worldwide. Mulberries are colorful berries that are eaten both fresh and dried. They're a good source of iron, vitamin C, and several plant compounds and have been linked to lower cholesterol, blood sugar, and cancer risk. Mulberries are the fruits of mulberry trees (Morus sp.) and are related to figs and breadfruit. The trees are traditionally grown for their leaves mainly in Asia and North America as they’re the only food that silkworms eat. They carry colorful berries most commonly black, white, or red that are often made into wine, fruit juice, tea, jam, or canned foods, but can also be dried and eaten as a snack. Mulberries have a decent amount of fiber. The present communication carries an attempt to evaluate the study of phytochemical physicochemical parameters e. g. successive extractive values in different solvents, ash values, alcohol, water, and hexane soluble extractives, moisture contents, loss of weight on drying at 105^oC, pH values of 1% and 10 % aqueous solutions, qualitative and quantitative mineral contents, and thin layer chromatography of different extracts. Besides, the qualitative and quantitative analysis of Phyto-constituents like alkaloids, total phenolics, tannins, sugar contents, crude fibers, proteins, and estimation of heavy metals, microbial loads, pesticide residues, and aflatoxins were also carried out which will be useful in the identification and control of the possible adulterations of the raw drug.

KEYWORDS: Standardization, Physicochemical properties, Mineral constituents, Quality control parameters, TLC.

INTRODUCTION:

The genus Morus is a very cold-hardy plant, a deciduous shrub or small tree that can grow up to 7.5 meters tall, Prefers warm well-drained loamy soil in a sunny position, widely distributed in Asia, Europe, North America, and South America, and Africa, and is cultivated extensively in eastern, central, and southern Asia for silk production. Mulberry is found in temperate and sub-tropical regions of the northern hemisphere, as well as in the tropics of the southern hemisphere because it can grow in a wide variety of climatic, topographical, and soil conditions. They spread throughout all regions from the tropics to the sub-arctic and from sea level to altitudes as high as 4000 m. These are grown at considerably higher altitudes in the Himalaya-Hindu Kush region, while in Pakistan they are widely cultivated in northern regions^{1- 6}.

Four or five species occur in India. Few of the Morus species are valued for their foliage, which constitutes the chief feed for mulberry silkworms. Leaves are very variable, ovate or broadly ovate, serrate or crenate-serrate often deeply lobed. Flowers are inconspicuous, greenish, male spikes-lax, flowered, broadly cylindrical, or ovoid. Female spikes-ovoid, pedunculate. Fruits are syncarp and consist of many drips enclosed in freshly perianth white to pinkish color. A monoecious, occasionally dioecious shrub or moderate-sized tree with a fairly cylindrical straight bole up to 3m in height and 1.8m in girth, mulberry is grown extensively for leaves used for rearing silk worms^7-8. Any pruning should only be carried out in the winter when the plant is fully dormant because mulberries bleed badly when cut. Ideally, prune only badly placed branches and dead wood ⁹. Plants in this genus are notably resistant to honey fungi¹⁰. The fruit is aromatic, cooling and laxative. Its use allays thirst and it is of help in the treatment of fevers. The bark is anthelmintic and purgative. A decoction of the leaves is used as a gargle in treating inflammation of the vocal cords. The root is anthelmintic and astringent ¹¹. A paste of the bark is used in the treatment of gingivitis ¹². The root bark is diuretic and pectoral. A decoction is used in the treatment of 'hot' pulmonary coughing, asthma, excessive sputum production, oedematous face, and difficult urination¹³. The bark fibers are used for making paper ¹⁴. Extracts of various parts of the plant are used as ingredients in commercial cosmetic preparations such as antiseborrheic, antimicrobials, antioxidants, astringents, emollients, and hair conditioners ¹⁵. The drug has antioxidant, Neuro-protective, Antitumor, and Immuno-modulatory activities¹⁶.

Keeping in view the great importance of this herb, an attempt has been carried out to establish its characteristics and scientific standards which can be employed as suitable quality control measures to ensure its safety and efficacy of it.

MATERIALS AND METHODS:

The fresh drug Morus indica Linn (fruits) was purchased from the local market, in Delhi. The fruits were transported to the laboratory in glass jars and held at room temperature in the laboratory. The drug was authenticated by the botanist Mrs. KiranNegi A. R. O. (Pharmacognosy). Fruits were dried at room temperature first and later these were dried in the oven at 69⁰C for 72 hours. Finally, this dried material was crushed and made into fine powder by a grinding machine for analysis. The sample was preserved as a specimen in the laboratory, “Drug Standardization Research Unit New Delhi”.

Determination of Alcohol and Water Soluble Extractives

Five grams of drug powder were placed in a glass-stopper conical flask and macerated with 100ml water for 6 hours with frequent shaking and then allowed to stand for the next 18 hours. The filtrate was then collected rapidly through a dry filter. 25ml of the filtrate was transferred to a previously weighed and tarred flat-bottom Petri dish and evaporated to dryness in a water bath. The residual material was dried at 105⁰C for 6 hours and cooled in a desiccator for 30 minutes before weighing. The percentage of the water-soluble matter was calculated with reference to the amount of the drug. The percentage of alcohol-soluble content was determined as mentioned above by using alcohol in place of water¹⁷.

Successive Extractive Values:

The successive extraction of the drug in three different solvents was carried out as per increasing order of their polarities viz. petroleum ether (40-60⁰C), chloroform, and alcohol by Soxhlet apparatus for six hours in a water bath. The contents so obtained were filtered by using filter paper. After evaporation of the solvents of filtrate in a water bath, the extractive values were determined with reference to the weight of the drug (% w/w). The procedure was repeated three times and the mean value for each extract was calculated¹⁸.

pH values of 1% and 10% aqueous solutions:

One gram of the drug was mixed in accurately measured 100 ml of distilled water and filtered and the pH value was measured with a pH digital meter. This procedure was repeated three times. The mean value and standard error were calculated. Similarly, for 10% aqueous solutions, ten grams of the drug was mixed in accurately measured 100ml of distilled water, filtered and pH measured with a pH digital meter by following the same procedure. The mean value and standard error were calculated¹⁸.

Loss of weight on drying at 105°C:

Two grams of the powdered drug were taken and spread uniformly and thinly in a shallow Petri dish. It was heated at a regulated temperature of 105⁰C, cooled in a desiccator, and weighed. The process was repeated several times till two consecutive weights were constant. The percent loss in weight was calculated with respect to the initial weight¹⁸.

Determination of crude fibers:

The known amount of the drug was exhausted first in 50 ml of diethyl ether by boiling for 30 minutes in the water bath to remove fats and waxes. 200ml of boiling sulphuric acid was added to the ether-exhausted drug in a 500ml flask and the flask was connected to a reflux condenser. The mixture was heated to boil for 30 minutes. Then it was filtered through filter paper and the residue was washed on the filter with boiling water till the filtrate lost its acidic character. The residue was rinsed back into the flask with 200 ml boiling sodium hydroxide solution and allowed to boil for 30 minutes. After boiling, the mixture was filtered through a tared filter, and the residue was washed with boiling water till it was neutral. The filtrate was dried at 110⁰C until the constant weight(X). The dried residue was incinerated and the ash was weighed(Y). Hence, the crude fiber content (X-Y) was obtained¹⁹.

The percentage of crude fiber (CF) is computed as

Weight of insoluble matter − Weight of ash

CF (%) = ------------------------------------------- X100

Weight of sample

= (B−C) /A X 100 (%)

Ash values:

Ash value of the drug is an inorganic residue remaining after incineration or all the organic matter has been burnt away from the drug. The method of total ash, acid-insoluble ash, and water-soluble ash are given as follows.

Total ash:

Two grams of the air-dried drug were incinerated in a silica dish at a temperature not exceeding 550⁰C until free from carbon, cooled, and weighed, and the percentage was calculated with reference to the air-dried drug (20).

Acid insoluble ash:

The total ash obtained in the previous experiment was boiled with 25ml of dilute hydrochloric acid for 5 minutes. The insoluble matter was collected on an ashless filter paper, washed with hot water and ignited at a temperature not exceeding 550⁰C, and weighed after cooling. The percentage of acid-insoluble ash was calculated with reference to the air-dried drug²¹.

Water soluble ash:

The total ash obtained was boiled with 25ml of distilled water for 5 minutes. The insoluble matter was collected on an ashless filter paper, washed with hot water, and ignited. The weight of insoluble ash was subtracted from the weight of the total ash, giving the weight of the water-soluble ash. The percentage of water-soluble ash was calculated with reference to the air-dried drug²¹.

Titratable Acidity:

The titratable acidity was determined by the titrimetric method ²². The sample of mulberry juice was weighed as 10g and 0.5ml phenolphthalein indicator was added and titrated against 0.1N NaOH. The endpoint was the appearance of a faint pink color. The volume of 0.1N NaOH used was recorded. The total acidity was calculated as

% Acidity = Volume of 0.1N NaOH (ml) × 0.064

The result is expressed as a percentage of citric acid (gram citric acid/100g of drug).

Total Protein:

Total protein content was analyzed using Kjeldhal’s method ²³. Weigh out the known amount of the drug and transfer it to a Kjeldahl digestion flask. Add the 8-gram catalyst mixture (96% anhydrous sodium sulfate, 3.5% copper sulfate, 0.5% selenium dioxide) and 20 ml of concentrated sulphuric acid. Heat the flask gently in an inclined position. When the initial frothing has ceased, fit a loose pear stopper in the top of the flask and then heat more strongly, so that the liquid boils at a moderate. Shake the flask from time to time and continue the heating for one hour after the liquid has become clear. Cool somewhat, wash the digest into the distilling flask with 400 ml of “ammonia-free” water, and add a large piece of granulated zinc. To the receiving flask add 50 ml of boric acid solution (2%) and screen methyl red indicator. Make the diluted digested alkaline with 50% sodium hydroxide solution and distill the ammonia into the boric acid solution. After about 300 ml has been distilled wash down the condenser and delivery tube into the receiver. Titrate the distillate with 0.1N sulphuric acid. The blank should not exceed 0.5 ml of 0.1N sulphuric acid. Calculate the percentage of nitrogen in the sample as given following way.

1ml 0.1N Sulphuric acid = 0.0014 gram of nitrogen

Further, the crude protein figure can be calculated using an appropriate factor: N X 6.25 (also the general factor)

Reducing and Non- reducing sugars:

Dextrose solution was prepared by dissolving 1.25grams in 250ml and titrating it with Fehling solution to find the dextrose reading. A 5-gram drug of dried fruit powder of mulberry was taken and 100ml warm water was added, stirred well, and filtered. The filtrate was then made up to 250ml in a volumetric flask (Solution A). Take out 50 ml of the solution into a conical flask, add 30ml of dilute HCl, and boiled for 5 min. The solution was cooled and add saturated lead acetate solution till it precipitates completely and filtered the whole content. Now the filtrate is neutralized to phenolphthalein with 10% NaOH and made up to 250ml (Solution B). About 25ml of Fehling's solution was taken in a conical flask and 4 drops of 1% methylene blue were added to it and boiled into separate flasks and boiled heating mental and already prepared sugar solutions A and B taken in the burette were titrated against Fehling's solution taken until the blue color disappears and note down the readings of solutions A and B. Percentage of total sugar and reducing sugar was calculated and nonreducing sugar can be estimated as the difference between the total sugar and the reducing sugar content from the following formula²⁴.

Non reducing sugar = (Total sugar - Reducing sugar) x 0.95

Total Lipids:

A 5-gram drug was homogenized using a mixture of chloroform and methanol in the ratio of 2:1 and made up to 25ml filtrate. The filtrate was poured into a separating funnel. Water, chloroform, and sodium carbonate were added, shaken vigorously, and kept for separation. Eluted the bottom layer of liquid into a pre-weighed Petri dish. The Petri dish was then kept in an oven to dry. The content was weighed to get the amount of lipid ²⁵.

Chemical Studies:

The detailed chemical analysis of the fruit parts of Morus indica Linn was carried out in the laboratory, Drug Standardization Research Unit situated in New Delhi. The studies included the determination of organoleptic properties, Physico-chemical and phytochemical studies with other quality control parameters as described below.

RESULTS:

Morphological characters:

The drug is purple or dark purple to black with an aromatic odor, sticky and acrid with a sweetish taste.

Chemical analysis:

Physico and Phyto-chemical Standards of Morus indica Linn (Fruits part):

Quantitative analysis of Physico and Phyto-chemical has been given in the following table.

Table no.1: Physico/phytochemical parameters

S. No.	Parameters	Results%	Mean +SD (%)
1.	Loss in weight on drying at 105°C	8.50 - 9.50	9.5+1.00
2.	Total ash	2.50 -3.10	2.86+0.36
3.	Acid insoluble ash	0.50 -0.75	0.60+0.10
4.	Water soluble extractive (%)	52.5 - 54.64	53.50+1.05
5.	Alcohol soluble extractive	51.4 - 53.25	52.90+1.50
6.	Hexane soluble extractive	2.20 - 2.54	2.24+0.04
7.	Successive extractive values (a) Petroleum ether (b) Chloroform (c) Ethanol	3.35 - 4.25 5.70 - 6.25 39.50 - 40.20	3.80+0.45 5.97+0.28 39.85+0.35
8.	pH Values (a) pH of 1 % aqueous solution (b) pH of 10 % aqueous solution	4.25,4.25, 4,25 4.75, 4.75, 4.75	4.25+0.00 4.75+0.00
9.	Sugar estimations (a) Total sugar (b) Reducing sugar (c) Non-reducing sugar	26.50 - 27.50 16.50 - 17.80 10.50 - 9.50	26.90+1.40 17.20+0.76 9.83+0.30
10.	Alkaloids	0.60 - 0.80	0.70+0.10
11.	Total phenolics	2.25- 3.10	2.62+0.37
12.	Tannins	0.85-0.98	0.92+0.07
13	Moisture contents	79 .80- 81.89	80.85+2.50
14	Lipid	1.80- 2.20	2.02+0.09
15	Protiens	1.60-1.85	1.73+0.07
16	Total carbohydrates	11.5-12.0	11.75+1.58
17	Titratable acidity	1.20-1.90	1.55+0.50
18	Calorific value (KCal /100 gram)	72.95	74.05+3.50
19.	Crude fibre	1.95-2.85	2.40+0.85

Mineral analysis: The dried fruits part powder of Morus indica Linn was ignited in the muffle furnace at 550⁰C. The ash was collected and subjected to a chemical test given under (26).

Qualitative:

Sodium (+)

Potassium (+)

Calcium (+)

Magnissium (+)

Iron (+)

Zinc (+)

Quantitative (%):

Sodium (0.38)

Potassium (1.15)

Calcium (0.75)

Magnesium (0.30)

Iron (0.10)

Zinc (0.03)

Organic constituents:

Different extracts of the drug were subjected to study for the organic analysis (27) and the qualitative analysis of phytoconstituents is reported in the following table no. 2.

Table no. 2: Qualitative Phyto-constituents analysis in Morus indica Linn

Chemical Constituents	Test / Reagent	Result
Glycoside	Liebermann test	+ve
Sterol	Salkowaski Reaction Liebermann-Burchard’s test	+ve
Phenolics	Phenol	+ve
Tannins	Ferric chloride test	+ve
Resins	Acetic Anhydride test	-ive
Amino acids	Ninhydrin Solution	+ve
Saponin	Sodium bicarbonate solution	-ive
Protein	Xanthoproteic test Biuret’s test	+ive
Flavonoids	Magnesium Chips test	+ive
Alkaloids	Mayer’s reagent	+ive
Carbohydrates	Fehling’s solution	+ive

Indications: (-ve) Absent and (+ve) Presence of constituents

Quality control parameters:

Microbial load, Heavy metals, Aflatoxins, and Pesticidal residue: In order to ensure the quality of the drug, modern techniques and standard methods were adopted. The parameters such as microbial load and heavy metals were carried out as per WHO guidelines. Aflatoxins and pesticide residues are carried out by standard methods. The heavy metals viz. lead, cadmium mercury, and arsenic as well as aflatoxins B1, B2, G1, and G2 and pesticide residues were not detected while analysis of microbial load shows that Total Fungal count (TFC) and Total Bacterial load (TBL) were within the permissible limit as stated by WHO while Escherichia coli, Salmonella spp, and Staphylococcus aureus were found to be absent (28-30).

Table no. 3: Estimations of Heavy Metals in Morusindica Linn

S. No	Name of Element analyzed	WHO and FDA limits of detection	Results
1.	Mercury	1 ppm	Not detected
2.	Lead	10 ppm	Not detected
3.	Cadmium	0.3 ppm	Not detected
4.	Arsenic	3 ppm	Not detected

Table no. 4: Analysis of Microbial Load

S. No	Parameter analyzed	Result	WHO limit
1.	Total Bacterial Count	Less than 1CFU/gm	10⁵ CFU/gm
2.	Total Fungal Count	Less than 1CFU/gm	10³ CFU/gm
3.	Enterobacteriaceae	Absent	10³ CFU/gm
4.	Salmonella	Absent	Nil
5.	Staphylococcus aureus	Absent	Nil
6.	Escherichia coli	Absent	Nil

Table no. 5: Estimation of Aflatoxin

S. No	Aflatoxin	Result	WHO limit (ppm)
1.	B1	Not detected	0.5
2.	B2	Not detected	0.1
3.	G1	Not detected	0.5
4.	G2	Not detected	0.1

Table no. 6: Analysis of Pesticide Residues

S. No	Pesticide Residues	Result	Limit
1	Alachor	Not Detected	LOQ : 0.01
2	Aldrin	Not Detected	LOQ : 0.01
3	Azinphos - methyl	Not Detected	LOQ : 0.01
4	Chlordane (cis and trans)	Not Detected	LOQ : 0.01
5	Chlorfenvinphos	Not Detected	LOQ : 0.01
6	Chlorpyrifos	Not Detected	0.064
7	Chlorpyrifos-methyl	Not Detected	LOQ : 0.01
8	Cypermethrin	Not Detected	LOQ : 0.01
9	DDT	Not Detected	LOQ : 0.01
10	Deltamethrin	Not Detected	LOQ : 0.01
11	Diazinon	Not Detected	LOQ : 0.01
12	Dichlorvos	Not Detected	LOQ : 0.01
13	Dimethoate	Not Detected	LOQ : 0.01
14	Dieldrin	Not Detected	LOQ : 0.01
15	Endosulphan	Not Detected	LOQ : 0.01
16	Endrin	Not Detected	LOQ : 0.01
17	Ethion	Not Detected	LOQ : 0.01
18	Fenitrothion	Not Detected	LOQ : 0.01
19	Fenvalerate	Not Detected	LOQ : 0.01
20	Heptachlor	Not Detected	LOQ : 0.01
21	Hexacholobenzene	Not Detected	LOQ : 0.01
22	Lindane (gamma HCH)	Not Detected	LOQ : 0.01
23	Malathion	Not Detected	LOQ : 0.01
24	Parathion	Not Detected	LOQ : 0.01
25	Parathion-methyl	Not Detected	LOQ : 0.01
26	Permethrin	Not Detected	LOQ : 0.01
27	Phosalone	Not Detected	LOQ : 0.01
28	Primiphos methyl	Not Detected	LOQ : 0.01

Indications: LOQ (Limit of quantification)

Chromatographic estimation:

Dried powdered drug (2 g) was extracted with ethanol, (25 ml) and chloroform (25 ml) under reflux conditions in a water bath for about 30 minutes and filtered. The filtrate was concentrated to 5 ml to carry out TLC. Extracts of ethanol and chloroform so obtained from the drug were spotted on two separated pre-coated silica gel 60 F254 aluminum plates using Toluene: Ethyl acetate: Acetic Acid (8:1.5:0.5) and Toluene: Chloroform: Acetic acid (8.5:1:0.5) respectively as a mobile phase. These were sprayed with 5 % ethanolic - Sulphuric acid and 1% ethanolic vanillin sulphuric acid respectively followed by heating at 105⁰C for 10 minutes in an oven (31-32). The ethanolic extract showed five spots at Rf 0.16 (Brownish Grey), 0.24 (Light Brown), 0.60 (Grey), 0.71 (Dark Brown), and 0.80 (Greyish Brown) with the Rf values 0.18 (Light brown), 0.51 (Greyish brown) at 366 nm while chloroform extract showed three spots at 0.30 (brown), 0.52 (greyish blue), and 0.61(grey) with the Rf values 0.52 (Light blue) at 254 nm as shown in table no. 7.

DISCUSSION:

Phytochemical screening of the secondary metabolites present in the drug revealed that it contains alkaloids, carbohydrates, glycosides, steroids, phenolics, tannins, proteins, flavonoids, and amino acids (Table 2). These chemical compounds may be responsible for the therapeutic efficacy of that drug. Organoleptic characteristics of the drug indicate that it is purple with a characteristic odor, sticky in nature, and sweet in taste. The mean percentage of total ash and acid insoluble ash was found to be 2.86, 0.60; whereas alcohol, water and hexane soluble extractives were found to be 52.90, 53.50, and 2.24 respectively. The percentage of moisture content was 80.85 while Loss of weight on drying at 105⁰C was found to be 9.50 gm%. The pH of the 1% aqueous solution was observed as 4.25 and the 10% aqueous solution was observed as 4.75. The solubility and extractive values sometimes are used as an index of purity for formulations. Therefore, for establishing the standards of any drug, these extractive values and solubility play an important role as the adulterated or exhausted drug material will give different values rather than the extractive percentage of the genuine. The loss of weight on drying on heating tells us about the moisture contents of the test drug, which determines the release of active ingredients as well as their chemical, physical, microbial, shelf-life properties and adulterations. The physicochemical Parameters data as given in table 1 are expressed as mean values of the three readings calculated. The pH values of all three samples were found to be acidic. Estimation of ash values is an important criterion for judging the impurities along with the identity and quality of drugs because adulteration of herbal drugs with unwanted materials like earthy matter results in a higher ash percentage. The study was extended for quantitative estimation of phytoconstituents, e.g. phenolics (2.62%), tannins (0.92%), alkaloids (0.70%), total lipids (2.02%), crude fibers (2.40%), Titratable acidity (1.55%), and proteins (1.73%) as presented % mean values of all the three samples in Table 1. The results of the total bacterial load and total fungal count of the microbial studies were within the permissible limits and the other parameters were found to be absent in the drug. The analysis of aflatoxins and heavy metal analysis showed that the drug was free from any contaminations. In quality control parameters, the bacterial load and total fungal count of microbial studies, aflatoxin, heavy metals, and pesticide residues were either within the permissible limits or found to be absent as shown in Tables 3, 4, 5 and 6 respectively. At the same time, bacteria Enterobacteriaceae, Salmonella spp, Escherichia coli, and Staphylococcus aureus were found to be absent which shows that the drug is not the carrier of these organisms. For the chromatographic studies, chloroform and ethanol extracts were prepared separately to carry out thin-layer chromatography by using different solvent systems, and their Rf values were reported as shown in Table no.7.

CONCLUSION:

The drug under study was subjected to physical parameters like morphological characters, and physicochemical analysis, with the other parameters such as phytochemical screening, TLC study, and quality control parameters of a drug such as heavy metal analysis, aflatoxins contamination analysis done and found absent; the microbial load was found within the permissible limits of WHO guidelines. TLC analysis was employed with respect to standardization and to separate the compounds which can be isolated for further studies. Considering the importance of the herb as a potential medicine, important analytical data have been reported here related to investigations of its physicochemical, phytochemical, mineral, and safety profile of it. These values of analytical parameters and TLC profiles together may be used for quality evaluation or as reference standards not only as an individual but also to be followed in the traditional production of any compound formulation in which this herb is added as an ingredient.

ACKNOWLEDGMENTS:

The authors are thankful to the Director General Central Council for Research in Unani Medicine, for the encouragement and support. The authors are also thankful to DSRI Ghaziabad for providing the necessary assistance.

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DOI: 10.52711/2231-5659.2023.00002

S. No.	Extracts	Solvent systems	No. of spots after spraying reagent	Rf values of spots observed under UV and after treatment with spraying reagent
S. No.	Extracts	Solvent systems	No. of spots after spraying reagent	254 nm	366 nm	Spray / Treatment of Visualizing agents
1.	Ethanolic extract	Toluene: Ethyl acetate: Acetic Acid (8: 1.5: 0.5)	5	None	0.18 (Light brown), 0.51 (Greyish brown)	5% Ethanolic sulphuric acid 0.16 (Brownish Grey), 0.24 (Light Brown), 0.60 (Grey), 0.71 (Dark Brown), and 0.80 (Greyish Brown)
2.	Chloroform extract	Toluene: Chloroform: Acetic Acid (8.5: 1: 0.5)	3	0.52 (Light blue)	None	5% Ethanolic Vaniline sulphuric acid 0.30 (Brown), 0.52 (Greyish blue), 0.61(Grey)