INTRODUCTION:

The chemical constituents of the medicinal plants, particularly the secondary metabolites have pronounced pharmacological actions on animal system and organs. Several bioactive compounds were isolated from the plants sources such as digoxin, reserpine, taxol, and vincristine, quercetin etc¹that has different pharmacological and chemical nutritional properties. The discovery of a novel chemical component from medicinal plant may form the basis of development of various therapeutic agents with better activity. The genus Hibiscus is widely distributed over Korea, china, India, and Siberia. The dried root of Hibiscus syriacus L. are used as a fork medicine in the orient² for the cure of hematochezia, dysentery, obstruction due to wind phlegm, and vomiting of food³.

Hibiscus syriacus L. (Rose-of-Sharon) is valued for large flowers produced in summer when few other shrubs bloom. It is useful as a garden accent due to its strict, upright habit. The open, loose branches and light green leaves make Rose-of-Sharon ideally suited to formal or informal plantings, and with a little pruning makes an attractive, small specimen tree.

The plant grows in sun or partial shade and in any soil. Rose-of-Sharon grows 8 to 10 feet tall and spreads 4 to 10 feet. The growth rate ranges from slow to moderate, and transplanting is easy. Several roots are usually located just beneath the soil surface. However, pruning is usually not required since the plant grows slowly and keeps a tight upright form⁴. Here we report the chemical constituents present in the leaves extract by chromatographic and spectrophotometric analysis and established the presence of triterpenoids as well as flavonoids⁵.

MATERIALS AND METHODS:

Selection of plant

Drug discovery from medicinal plants has evolved to include numerous fields of inquiry and various methods of analysis. The process typically begins with a botanist, ethanopharmacologist who identifies the plant of interest.Collection may involve species with known biological activity. Based on intensive literature survey.Hibiscus Syriacus was selected for present study.

Collection of plant materials

The leaves of Hibiscus syriacus L. was collected from the month of august-September from the Garden of Jawaharlal Institute of Technology and G. R. Y. Institute of Pharmacy Vidya Vihar Borawan district Khargone western nimar region of Madhya Pradesh

Authentication of plant

The plant Hibiscus syriacus L. was identified and authenticated by Dr S.K. Mahajan, Botanist from Government College, Khargone Madhaya Pradesh. The herbarium of the plant specimens were prepared and deposited in the Department of Pharmacognosy, G. R. Y. Institute of Pharmacy vidya vihar Borawan district Khargone madhaya Pradesh, India, under voucher no. G.R.Y.I.P. 43.

Preparation of extract

Extraction of organic component

The leaves were initially separated from the main plants body and rinsed with distilled water, dry in shade then homogenized into fine powder and finally stored in air tight bottles. It was then passed through the 40 mesh sieve. A dried and powered plant material was defatted firstly to remove fatty material. For this purpose 1000 gm of weighed powered plant of Hibiscus syriacus L. was packed in Soxhlet apparatus and extracted with petroleum ether at 60-80°c for 36 hrs and completion of extraction was confirmed by discoloration of the solvent. The marc was removed and dried then it was subjected to continuous hot extraction with organic solvent like benzene, chloroform, and methanol in soxhlet apparatus for 36 hrs and completion of extraction was confirmed by discoloration of the solvent. After complete extraction of plant materials the solvent was evaporated for concentration of extracts. The petroleum ether, benzene, chloroform, and methanolic leaves extract of Hibiscus syriacus L. yielded greenish brown, green, brown and deep blue semi solid residue, then it were filtered with the help of muslin cloth. The supernatant was collected and the solvent was evaporated by solvent distillation apparatus and concentrate the extract in reduce pressure⁴.

Extraction of aqueous component

The marc was removed and dried then it was subjected to boil with 2 liter of water for 24 hrs, and then filtered through muslin cloth, and extract was evaporated by solvent distillation apparatus⁴.

Phytochemical analysis of extract

The methods described by Harborne (1978) with slight modifications were used to test for the presence of the active ingredients in the test sample. The methanolic extract and various chromatographic fraction were subjected to preliminary phytochemical screening for detection of various phytoconstituents^{6, 7}.

Fractionation of plant material

The methanolic extract was filtered and the filtrate was concentrated in vaccum. The residue partioned between n-hexane and water and then water layer was extracted with chloroform, successively. The chloroform layer was concentrated under reduced pressure^8,9 and kept in airtight containers for further analysis¹⁰.

Solvent optimization

The influence of various organic solvents on the retention of secondary metabolite activity was related to solvent properties like the polarity and the molecular size (as expressed by the molecular weight or molar volume). High activity retention was favored by a low polarity in combination with a high molecular weight. The solubility parameter also proved useful to describe the capacity of various organic solvents. This facilitated the optimization of the solvent polarity.

Isolation and Identification of Flavonoid by Column Chromatography

The chloroform soluble materials of methanolic extract were fractionated by silica gel column chromaroghaphy. The column was packed with silica gel and soaked with a ratio of benzene: ethyl acetate: n-butanol:n-hexane:acetic acid(50:25:15:10) for 12 hours. The column was eluted with same solvent mixture. Total 2000ml volume was collected in the mentined solvent system. About 5g of methanol fraction was chromatographed over silica gel and elution was carried out by solvent system using isocratic elution method.

1. Adsorbent : Silica gel (60-120 mesh)

2. Activation: 110ºC for 1 hour.

3. Length of the column : 96cm

4. Diameter : Outer 2.8 cm. Inner 2.1 cm

5. Length of the adsorbent : 60 cm

6. Rate of elution: 10-15 drops/min.

7. Volume of Elute collected 10ml each.

8. column dimention: 1:30

9. Type of Elution: Isocratic elution¹¹.

Preparation of Sample for Column

5 gm of methanol fraction was dissolved in 20ml of methanol, mixed with 2gm of silica gel (60-120 mesh), and dried in vacuum oven at 45 º C. The material was then transferred to the column.

Phytochemical test for isolated flavonoid

1. Zn-Hcl reduction test: To the test solution a mixture of zinc dust was added, few drops of concentrated Hcl was added dropwise along the side of the tube, which showed red colour indicating flvanoids present.

2. Ferric – Chloride test: To the test solution, freshly prepared ferric chloride solution was added, which showed bluish green to black colour¹².

RESULT AND DISCUSSION:

Preliminary phytochemical screening

Preliminary phytochemical screening for Hibiscus syriacus L. leaves (table 1) revealed the presence of carbohydrate, glycosides, steroids, proteins, flavonoids, tannins and alkaloids.

Optimization of chromatographic condition

All chromatographic condition was illustrated in table2. The chromatographic detection was performed using UV detection chamber. Chromatographic condition were optimized by changing the mobile phase composition and buffers used in the mobile phase.The optimized mobile phase was determined as mixture of Benzene: Ethyl acetate, n-Butanol: Acetic Acid (50:25:15:10) in different proportion to achieve maximum seperation and sensitivity. Parameter such as mobile phase composition, wavelengh of detection, column dimension, column temperature, pH of the mobile phase were optimized.Several proportion of buffer and solvent were evaluated in order to obtain suitable composition of mobile phase.

Table1. Phytochemical screening of leaves extract of Hibiscus syriacus L.

S.No.	Phytochemical constituents	Pet.ether extract	Benzene extract	Chloroform extract	Methanol extract	Water extract
1	Carbohydrates	-	+	+	+	+
2	glycosides	-	-	-	+	+
3	Steroids	+	+	+	-	-
4	Proteins	-	-	+	+	-
5	Flavonoids	-	-	-	+	+
6	Tannins	+	+	+	+	+
7	Alkaloids	-	-	-	-	+

Table2. Solvent optimization of chromatographic condition by TLC

S.No.	Chloroform (ml)	Ethylacetate (ml)	Acetone (ml)	n-Butanol (ml)	Acetic acid (ml)	Benzene (ml)	Formic acid (ml)	Water (ml)	Remarks
1.	4	4	-	2	0.5	-	-	-	OK
2.	4	6	-	2	0.5	-	-	-	OK
3.	3	7	-	2	0.5	-	-	-	-
4.	2	8	-	2	0.5	-	-	-	-
5.	5	3	-	2	1	-	-	-	OK
6.	6	2	-	2	1	-	-	-	OK
7.	-	4	-	4	1.2	4	-	-	-
8.	-	3	-	2	1	5.5	-	-	-
9.	-	3	-	2	1	7	-	-	-
10.	-	3	-	2	1	7	-	-	-
11.	75	-	16.5	-	-	-	8.5	-	OK
12.	-	100	-	-	11	-	11	27	-
13.	60	40	-	-	-	-	-	-	-
14.	-	2.5	-	1.7	0.80	5	-	-	-
15.	-	-	40	-	10	-	-	50	-
16.	6	3	-	-	1	-	-	-	-
17.	-	3	-	-	1	6	-	-	-
18.	-	5	-	3	2	10	-	-	-

Table3. Isocratic elution of Flavonoid by Column chromatography

Fraction	Solvents and it’s ratio	Colour of the elute	TLC solvents ratio	No. of spots and Rf. Values	Yield in mg
01-16	Benzene: Ethyl acetate, n-Butanol: Acetic Acid (50:25:15:10)	No colour	Benzene: Ethyl acetate, n-Butanol: Acetic Acid (50:25:15:10)	No spot	00
17-23		Dark Red colour		Single spot (0.9)	10
25-39		Light blue colour		Single spot (0.7)	48
40-60		No colour		No spot	30
61-70		No colour		No spot	20
71-120		Light blue colour		Double spot (0.8), (0.6)	100
121-140		No colour		No spot	30
141-200		Light blue colour		Double spot (0.75), (0.4)	200
201-206		No colour		No spot	10

Table4. Phytochemical test for isolated flavonoids

S.No.	Fraction no.	Fraction code	Phytochemical test for isolated flavonoids
S.No.	Fraction no.	Fraction code	Zn-Hcl test	Ferric-Chloride test
1.	17-23	A	-	-
2.	25-39	B	+	+
3.	40-60	C	-	-
4.	61-70	D	-	-
5.	71-120	E	+	+
6.	121-140	F	-	-
7.	141-200	G	+	+
8.	201-206	H	-	-

Table5. Determination of λmax of fraction

S.No.	Fraction no.	Fraction code	Λmax (nm)
1.	B	25-39	285
2.	E	71-120	209
3.	G	141-200	214

Fig1. Determination of λmax of fraction code B Fig2. Determination of λmax of fraction code E

Fig3. Determination of λmax of fraction code G

Isocratic elution

Isocratic elution was carried out by using Benzene: Ethyl acetate, n-Butanol: Acetic Acid (50:25:15:10) in different proportion and total 206 fraction were collected. The elution rate was adjusted to 10-15ml/minute. Different fractions like 1-16, 17-23 25-39, 40-60, 61-70, 71-120, 121-140, 141-200, and 200-206 were eluted. TLC studies were carried out using Benzene: Ethyl acetate, n-Butanol: Acetic Acid (50:25:15:10) using UV chamber for all the fractions. The fractions 1-16 did not exhibit any spots. However, in between the 17-23 fractions exhibited single spot with Rf value 0.9 which showed red spot under u.v. light. The 25-39 fractions exhibited single spot with Rf value 0.7. Fractions 40-60 did not exhibit any spots, 61-70 fraction did not exhibit any spots, 71-120 fraction exhibited double spot with Rf value 0.8, and 0.6, 121-140 fractions did not exhibit any spots, and 141-200 fraction exhibited double spot with Rf value 0.75 and 0.4, 201-206 fractions did not exhibit any spots.

SUMMARY AND CONCLUSION:

Various fraction of methanolic extract obtaied by column chromatography was collected and were tested for presence of flavonoids. Fraction no. 25-39, 71-120 and 141-200 gave positive test for flavonoidswhich was further confermed by UV spectrophotometer. Hibiscus syriacus L. have shown activity like analgesic, antiinflammatory, antibacterial, hair growth promoting activity antioxidant activity because of this reason the plant was selected and plant extract was further analyzed for seperation of flavonoids constituent in the plant.

The fraction no. 25-39, 71-120 and 141-200 having flavonoids and this flavonoids will be seprated and will be tested for Biological activity.

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Received on 07.01.2015 Accepted on 18.02.2015

Asian J. Pharm. Tech. 2015; Vol. 5: Issue 1, Pg 8-12

DOI: 10.5958/2231-5713.2015.00002.1