INTRODUCTION:

Malva sylvestris L. (Malvaceae), commonly known as Gulkhair or Vilayti Kangani, is an erect, branched, woody perennial or biennial herb up to 120 cm high. It is native to Europe mainly to Italy; distributed in western Asia, North America, and temperate western Himalayas from Kashmir to Kumaon between 700-2700m, southern India, Siberia, Australia and China¹. All parts of the plant are rich in mucilage.

The herbs are antiseptic, demulcent, emollient and refrigerant and sore throat, chronic bronchitis, jaundice, enlarged spleen; strangury, urinary dischargers and scorpion string². The flowers and immature fruits are efficacious to cure whooping cough and are official in French and Swiss pharmacopoeias¹. Malvin², 8-hydroxy flavonoid glucronides³, malvidin- 3 - O - (6”- malonyl Glucoside) - 5 - O - glucoside⁴, terpenoids and phenolic derivatives⁵, acidic polysaccharides⁶, chlorophylls, carotenoides⁷ and malovne - A⁸ have been reported from the plant. Antiproliferative⁹, antioxidant^10-11, anti-microbial^12-13 and anti-complimentary¹⁴ activities and effects on the production of anti-egg albumin antibodies¹⁵have been studied. Due to such great medical values, the drug was selected for screening the anti-inflammatory activities against carrageenan-induced rat paw oedema test to find out the activity of the test drug.

Plant Material and Methods:

The four samples of plant materials of fruits of M. sylvestris L. for work were procured from different sources of the local market like Delhi, Aligarh, and Lucknow and one sample was freshly collected from cultivated sources and taken as a standard sample. All three market samples were matched with the collected fresh samples and also the samples with reference herbarium specimens preserved in the Drug Standardization Research Unit (CCRUM), Faculty of Science, Jamia Hamdard, Hamdard Nager, Nagar, New Delhi. Finally, the material & was identified by the Botanist Dr Raisuddin Ahmed. These were cleaned and dried authenticated under the shade in a dust-free environment and powdered with a mechanical grinder.

Preparation of Extract:

The fruit powder was air dried in shade for 78 hours and again over-dried for 72 hrs below 50⁰C. The dried powdered drug (100g) was extracted with water on an electric hot plate and with ethanol in a Soxhlet apparatus separately. The extracts were concentrated and dried using a rotary flash evaporator at 50⁰C to get a dark viscous mass. The powdered drug, petroleum ether, ethanol and the aqueous extracts were utilized for screening the anti-inflammatory activity

Fig. 1 Fruit parts of Malva sylvestris L

Animals :

Wistar strain albino rats (150-200g) of either sex maintained under standard conditions temperature 23+20⁰C, relative humidity: 55+10% and 12 hrs. light and dark cycle), were used for all the experiments, comprising five rats in each group. The rats were allowed standard laboratory feed and water ad libitum¹⁶.

Drugs:

Oedema was induced with carrageenan (0.1ml) in 1% suspension in CMC by injecting it into the sub-planar region of the right hind paw of each rat. The alcoholic extract (200mg/Kg) and standard drug indomethacin (20 mg/Kg) were dissolved in saline and administered orally to rats 30 minutes before the injection of carrageenan. Control rats received an equal volume of CMC. The volume of oedema was measured with a plethysmometer after two hours of carrageenan injection. A solution (90%, w/v) was prepared in normal saline and a test drug was given to the rats through the intraperitoneal route^17-20.

Statistical Analysis:

The values were expressed as the mean+SEM for each parameter. The significance was calculated by using the student t-test.

Toxicity Studies:

The rats were divided into control and test groups. The control groups received vehicles and the test groups received different suspensions of extracts 0. 1, 0.2, 0.5, 1.0, 5.0, 7.0, and 10.0/kg P.O and were for mortality till 48 hours.

Anti - Inflammatory Activity:

Carrageenan-Induced Rat Paw Oedema test:

Administration of carrageenan into the sub plantar region of rats produced oedema, which resulted in a significant increase in paw volume. Indomethacin, which was used as a standard at a dose of 20mg/kg, exhibited a marked reduction in increased oedema volume compared to that of animals in the control groups. The aqueous and ethanolic extracts at a dose of 100mg/Kg, and 200 mg/kg, respectively, showed significant anti-inflammatory activity as given in Table no. 1.

Table No. 1. Anti-inflammatory Activity of Different Extracts of M. sylvestris (Fruits)

S. No.

Groups

Treatment

(Dose) mg/Kg I.P.

The difference in paw volume after

1 Hr 2Hrs.

Anti-inflammatory

Effect (Mean %)

Normal Saline

(Control)

0.40 + 0.01 0.59 + 0.02

1 Hr. 2Hrs.

Indomethacin

(Standard)

0.30 + 0.02 0.29 + 0.03

25.00 50.84

(a)

(b)\

(c)

(d)

Test Groups

Powder as such

Pet. ether extract

Ethanol extract

Aqueous extract

500

100

200

100

0.60 + 0.02 0.72 + 0.01

0.31 + 0.02 0.63 + 0.22

0.27 + 0.01 0.26 + 0.02

0.15 + 0.01 0.20 + 0.02

-ive -ve

12.50 -ive

32.50 55.93

62.50 66.10

The significant reduction compared to control (P<0.001)

Table no. 4. Phytochemical screening of successive extracts of fresh samples

S. No	Phyto-constituents	Sample A			Sample B
S. No	Phyto-constituents	Pet Ether	Chloroform	Ethanol	Pet Ether	Chloroform	Ethanol
1	Steroids	+	+	+	-	+	+
2	Triterpenes	+	+	+	+	+	-
3	Glycosides	-	+	+	-	-	+
4	Phenolics	-	-	+	-	-	+
5	Tannins	-	-	+	-	-	+
6	Resins	+	+	+	+	+	-
7	Flavonoids	-	+	+	-	-	-
8	Saponins	-	+	+	-	+	+
9	Fixed Oils	+	-	-	+	-	-
10	Alkaloids	-	+	+	-	+	-

Continew Table 4

S. No	Phyto-constituents	Sample C			Sample D
S. No	Phyto-constituents	Pet Ether	Chloroform	Ethanol	Pet Ether	Chloroform	Ethanol
1	Steroids	-	+	+	-	+	+
2	Triterpenes	+	+	-	+	-	-
3	Glycosides	-	+	+	-	+	+
4	Phenolics	-	-	+	-	-	+
5	Tannins	-	-	+	-	-	+
6	Resins	+	+	-	+	-	+
7	Flavonoids	-	-	-	-	-	+
8	Saponins	-	+	+	-	-	-
9	Fixed Oils	+	-	-	+	-	-
10	Alkaloids	-	+	+	-	+	+

Indications: (+) Present, ( - ) Absent, A = Fresh Sample, B = Delhi Marke, C = Aligarh market, D = Lucknow market

Table no. 5: Physico-chemical constants

S. No	Physicochemical Constants (%)	Sample A	Sample B	Sample C	Sample D
1	Petroleum Ether	6.90 + 0.090	6.78 + 0.032	6.20 + 0.035	5.88 + 0.075
2	Chloroform Extract	4.80 + 0.180	4.59 + 0.015	3.95 + 0.04	3.98 + 0.035
3	Ethanol Extract	4.74 + 0.095	3.28 + 0.025	3.98 + 0.019	3.21 + 0.005
4	Alcohol Soluble Matter	12.00 +0.082	10.80 +0.053	9.85 + 0.030	9.60 + 0.030
5	Water Soluble Matter	22.64 +0.075	19.54 +0.065	18.08 +0.062	17.45 +0.025
6	Total Ash	9.95 + 0.028	10.25 +0.053	10.48 +0.040	11.08 + 0.05
7	Acid Insoluble Ash	4.45 + 0.019	5.25 + 0.39	5.40 + 0.018	5.80 + 0.04
8	Water Soluble Ash	0.591 +0.030	0.253 +0.012	0.38 + 0.050	0.30 + 0.036
9	Unsaponifiable Matter	3.95 + 0.12	4.3 + 0.15	5.8 + 0.09	7.8 + 0.152
10	Moisture Content	3.80 + 0.039	4.85 + 0.025	4.90 + 0.015	5.05 + 0.06
11	pH 10% AqSoln	5.82	5.81	5.82	5.81
12	pH 10% AqSoln	5.51	5.54	5.54	5.51
13	Tannin Content	0.015 +0.035	0.010 + 0.02	0.012 +0.012	0.009 + 0.04
14	Resin Content	7.80 + 0.034	6.99 + 0.050	6.15 + 0.049	5.93 + 0.029

Data indicates the means of 3 values + Standard error, A = Fresh Sample, B = Delhi Market, C = Aligarh market, D = Lucknow market

Oil analysis:

Petroleum ether extract (oil) was utilized to carry out some important parameters like specific gravity, saponification value, acid value, ester value and peroxide value as given in the following table.

Table no. 6: Characteristics of Oil of Malva sylvesteris Linn (Fruits):

Parameters	Sample A	Sample B	Sample C	Sample D
Specific gravity of Oil	1.498 +0.005	1.498 + 0.001	1.497 + 0.002	1.498 + 0.002
Saponification Value	203.4 + 0.005	203.8 + 0.543	203.4 + 0.158	209.9 + 0.187
Acid Value	12.52 + 0.010	12.78 + 0.289	14.88 + 0.180	16.90 + 0.30
Ester Value	190.98 + 0.013	192.00 + 0.244	192.6 + 0.078	191.6 + 0.157
Peroxide Value	7.39 + 0.038	7.59 + 0.05	9.60 + 0.08	13.98 + 0.020

Data indicates the means of 3 values + Standard error, A = Fresh Sample, B = Delhi Market

C = Aligarh market, D = Lucknow market

Quantitative determination of fatty acids composition:

By using the Soxhlet apparatus extraction of the powered drug was carried out in petroleum ether (bp 60-80⁰C), filtered, distilled off the petroleum ether and the extract (oil) was collected.

Preparation of Methyl Esters of Oil:

The methyl esters of fatty acids present in oil were prepared by the esterification method proposed by Morrison and Smith (1964) with slight modification. The oil (0.1ml) was taken in airtight capped glass tubes. 0.5 ml of sodium methoxide (prepared in methanol) was added. The tubes were capped and checked that they were airtight. The contents of the tubes were mixed thoroughly and heated in boiling water for 10 minutes. The tubes were cooled in cold water. Two drops of BF₃ (prepared in 14% methanol) were added to the solution in each tube, and then there were heated for 10 minutes. After cooling in cooled water, 5 ml of hexane was added to each tube and mixed thoroughly. After about 10 minutes the content of the tubes gets separated into two layers. The upper layer containing methyl esters was taken out, concentrated and kept in separate vials which were utilized for determining the fatty acids composition by GLC analysis.

For determination of fatty acids, the esters of standard samples of C_12, C_14, C_16, C_18.0, C_18.1,C_18.2,and C_18.3 (lauric, myristic, palmitic, stearic, oleic, linoleic and linolenic, respectively) were mixed in different proportions and injected by maintaining the temperature 220⁰C. The gas flow rate was 1 ml/min and a packed column with an FID detector was used. Data from the chromatograms were recorded. In continuation of it, by applying the same conditions, the test sample (methyl ester of petroleum ether extract of M. sylvestris fruit part) was injected to know the fatty acid Composition. Finally, the composition of fatty acids^26-28was reported by comparing the data of both the peak tables (standard and test samples). The test sample was also analyzed against the linseed oil ester and the composition of unsaturated fatty acids (C₂₀-C₂₂) was also reported. The comparison of the peak table for chromatograms of standard samples (C_12, C_14, C_16, C_18.0, C_18.1,C_18.2) with the peak table for chromatograms of test sample; the following data were found which are given in the table below.

Table no.8: Fatty acids Composition

S. no.	Name of fatty acids	Area per cent	Quantity (%)
1.	Lauric (C12)	1.4	1.87
2.	Myristic (C14)	4.5	6.04
3.	Palmitic (C16)	28.0	37.53
4.	Stearic (C18:0)	7.40	9.93
5.	Oleic (C18:1)	19.50	26.17
6	Linoleic (C18:2)	10.70	14.36
7.	Unsaturated (C20-C22) analyzed against standard linseed oil ester	3.00	4.03

Quantitative Elemental Analysis:

Total ash so obtained from the drug was used to prepare the solution and the Concentration of the elements was reported in part per million by using an Atomic Absorption spectrophotometer.

Table no. 9: Atomic Absorption Spectrophotometer Analysis

Element	PPM
Element	A	B	C	D
Iron	18	10.8	12.5	7.9
Calcium	1381	1221	1075	855
Magnesium	433	376	380	272
Sodium	36	32	28	10
Potassium	192	165	172	110
Lead	ND	0.25	1.5	4.5
Cadmium	ND	0.01	0.05	0.09
Mercury	ND	ND	0.02	0.02
Arsenic	ND	ND	0.03	0.05

A = Fresh Sample, B = Delhi Market, C = Aligarh market, D=Lucknow market, ND= Not detected

Chromatographic Study:

Successive extracts of petroleum ether, chloroform and ethanol were utilized to carry out the thin layer chromatography and the Rf values so obtained have been reported in the following table.

Table no. 10: Rf Values of Different Extracts

Rf Values of Different spots	Petroleum Ether Extract				Chloroform Extract				Ethanol Extract
	Sol Sys Pet Ether: CHCl₃ (19:1)				Sol Sys Pet Ether: CHCl₃ (19:1)				Sol Sys Pet Ether: CHCl₃ (19:1)
	Treatment 5 % H₂SO₄ in EtOAc				Treatment 5 % H₂SO₄ in EtOAc				Treatment 5 % H₂SO₄ in EtOAc
	A	B	C	D	A	B	C	D	A	B	C	D
	0.05	0.05	0.08	0.05	0.24	0.24	0.23	0.22	0.18	0.18	0.17	0.18
	0.20	0.20	0.21	0.23	0.34	0.34	0.3	--	0.30	0.30	0.31	--
	0.38	0.38	0.37	0.39	0.53	0.53	0.58	0.53	0.50	0.50	0.52	0.40
	0.70	0.70	0.68	0.65	0.78	0.78	0.76	0.79	0.90	0.90	0.86	0.91
	0.90	0.90	0.85	0.8	0.94	0.94	0.91	0.93	--	--	--	--

DISCUSSION:

The fruits part of Malva sylvestris Linn were selected based on their utility profile in the indigenous systems of medicine. The present study was undertaken to investigate the anti-inflammatory activity of different extracts with the phyto and physicochemical studies of the selected drug. The study revealed that among the crude powder, petroleum ether, ethanol and aqueous extracts, only ethanol and aqueous extracts of fruits parts of Malva sylvestris showed significant anti-inflammatory activity (P<0.001) against carrageenan-induced paw oedema in rats, while rest were found inactive. The plant containing steroidal lactones like Wthania somnifera exhibits anti-inflammatory effects²⁹. The fruits part of this plant has also reported six new steroidal lactones³⁰and showed significant anti-inflammatory activity. In phytochemical studies, qualitative analysis of successive solvent extracts showed the presence of organic constituent mucilage, glycosides, steroids, triterpenes, fixed oils saponins, proteins phenolics, tannins, resins and flavonoids. The chromatographic studies also confirmed the presence of these constituents. Proximate analysis showed the presence of moisture contents, total ash, acid insoluble ash, water soluble ash, alcohol soluble matters and water soluble matters. Acid soluble ash showed the presence of sodium, potassium, calcium, and magnesium. Results of quantitative determination of fatty acid composition and estimation of heavy metals like lead mercury cadmium arsenic by atomic absorption spectrophotometer were also reported. As far as qualitative analysis is concerned, it seems to be no difference between fresh and market samples. Whatever the differences may be, are therefore more quantitative than qualitative. Physico-chemical parameters results of the samples were showing somewhere greater variation or a somewhere slight difference in their values but the TLC profile of all the extracts of various samples are showing not much difference in Rf values with respect to the particular extract. The Rf values of only major spots were shown. However, the number of spots produced by each sample varied including the minor spots. This was because of naturally possible for the samples to come from different sources of collection, depending upon various environmental factors affecting the variability in crude drugs, and the age of the plants. By comparing the results of market samples with the freshly collected plant material obtained from the cultivated sources, it was found that the Lucknow market sample (Sample- D) was found to be of lesser quality (Fourth grade), while the Delhi market sample (Sample-B) was found to be of first grade.

CONCLUSION:

The present studies provide a basis for further detailed investigations on the therapeutic efficacy of the fruits Malva sylvestris for ascertaining their anti-inflammatory potential claimed in the indigenous systems of medicine. The analytical data of the Delhi sample (Sample- B) can be taken as standard for market samples in future because the data of this sample is quite equivalent to a sample-A which is fresh and of high quality. But in the market, this standard can’t be maintained, as the supply of drugs can’t be so frequent in all parts of the country, and if it will be maintained, it leads to a high cost due to an increase in freight charges but with one-year-old material, the frequent supply can be easily maintained and there will be no storage in the Indian market. Besides this, it is very clear that the drug should be stored in an airtight container and not for more than one year. Faulty techniques and long storage of the drug are not affecting only the oil but also the quality of the oil is badly influenced.

ACKNOWLEDGEMENTS:

The authors are thankful to the Director General Central Council for Research in Unani Medicine, and Vice Chancellor Jamia Hamdard, New Delhi for the encouragement and support. Thanks are also due to colleagues of the Drug Standardization Research Unit, Faculty of Science, Jamia Hamdard, New Delhi where this work was undertaken.

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Received on 22.08.2022 Modified on 05.09.2022

Res. J. Pharmacology and Pharmacodynamics.2022;14(4):219-224.

DOI: 10.52711/2321-5836.2022.00038