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RESEARCH ARTICLE

In- Vitro and in-Vivo Anti-Inflammatory Activities of Salvia hispanica and Linum usitatissium Seeds in Swiss Albino Rats

Vidyasabbani¹*, A. Ramesh¹, Snehalatha²_, Bhanu Rahul², Sriharitha², Sanjayvarma², Aparna²

¹Department of Pharmacology, Vishnu Institute of Pharmaceutical Education and Research, Narsapur, Medak, Telangana, India.

²Department of Pharmacology, Sitha Institute of Pharmaceutical Sciences, Rajivgandhi Nagar, Hyderabad. Telangana, India.

*Corresponding Author E-mail: vidya.s@viper.ac.in.

ABSTRACT:

Salvia hispanica called as Chia seeds family Lamiaceae and Linum usitatissium called as Flax seeds family Linaceae was evaluated for its invitro and invivo anti-anti inflammatory activities individually and combined in swiss albio rats. Phytochemical screening analysis of both seeds revealed the presence of flavanoids. The Fourier Transform Infrared Spectrophotometer (FTIR) analysis of chia and flax seeds confirmed the presence of amide, alcohols, phenols, alkanes, carboxylic acids, aldehydes, ketones, alkenes, primary amines, aromatics, esters, ethers, alkyl halides and aliphatic amines compounds, which showed major peaks. In-vivo anti-inflammatory activity was done on Swiss albino rats (150-250 gms) at two different doses (100 and 200 mg/kg body weight) by using 1% histamine induced paw edema test. In -vitro anti-inflammatory test was done by protein-denaturation and the human red blood cell (HRBC) membrane stabilization method. Data were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett’s t-test and the test of significance P value <0.05 was considered as the minimum level of significance. The paw volumes and percentages of inhibition of inflammation with chia and flax and standard drugs are reported and were observed that the seeds of chia and flax combination showed Synergistic action of inhibition at two doses compared to individual seeds. The inhibition was the highest at 3 h at 200 mg/kg dose which was slightly lower than Indomethacin effect. In the study of membrane stabilization activity of chia and flax combination at concentration range of 100-200 μg/ml protected significantly in a concentration dependent manner the erythrocyte membrane against lysis induced by hypotonic solution. Acetylsalicylic acid in the concentration of 100 μg/ml used as standard also offered significant (p<0.001) protection of RBC’s membrane against damaging effect induced by hypotonic solution. The present research concludes that seeds of Salvia hispanica and Linum usitatissium possess anti-inflammatory activity in dose dependant manner and also proved synergistic activity.

KEYWORDS: Anti-inflammatory, Salvia hispanica and Linum usitatissium, Protein-denaturation, Indomethacin, Phytochemical screening, Human red blood cell (HRBC), Membrane stabilization.

Niacin is an important B-complex vitamin found abundantly in chia, nearly more than double the amount in sesame seeds[4]. The seeds are incredibly rich sources of many essential minerals. Calcium, phosporous, iron, manganese, and magnesium are particularly concentrated in chia. Many of these minerals have a vital role in bone mineralization, red blood cell production, enzyme synthesis, as well as regulation of cardiac and muscle movement[5]. Linum usitatissium commonly called as Flax seeds are used for various purposes such as anticoagulant and ant platelet effects [6] Antihypertensive affects [7] Antilipidemic effects[8]Antioxidant effects [9]. In this report, we summarize the anti-inflammatory activities of the combined bi-herbal preparation made up of equal quantities of seeds of Salvia hispanica (Chia seeds) and Linum usitatissium (Flax seeds). Histamine -induced inflammation in the rodent paw presents a classic model of oedema formation and hyperalgesia. This model was used for the in vivo experiments. In vitro anti-inflammatory test by protein-denaturation and the human red blood cell (HRBC) membrane stabilization method.

MATERIALS And METHODS:

Collection of Drugs (Salvia hispanica Seeds and Linum usitatissimum Seeds):

Both the seeds were obtained from Spice Line Organic stores, Hyderabad

Drug treatments:

Salvia hispanica and Linum usitatissium seeds were administered orally as 1% Gum acacia suspension, in the doses of 100 and 200 mg/kg individually and combination. Control rats were treated with equal volume of vehicle 1ml of 1% Gum acacia suspension.

Chemicals:

Gum acacia, (SD fine chemicals), Disodium hydrogen phosphate, Mono sodium di hydrogen phosphate, Acacia, Hydrogen peroxide, Picric Acid, Tri sodium citrate, Aluminium trichloride. (SD fine chemicals.

Phytochemical Screening:

Phytochemical examinations were carried out the two plants as per the standard methods [10,11and12] described in Table 1

Animals:

Swiss albino wistar rats were used for study. Wistar rats (150--250 g) procured from the National Institute of Nutrition, Hyderabad, were maintained under standard environmental conditions. Animals had free access to feed (Hindustan Liver, Bangalore) and tap water adlibitum during the quarantine period. All procedures compiled with the norms of the animal ethics committee of our university. Supervision of Experimental Animals (CPCSEA). The animals were maintained in well ventilated room temperature with natural 12 ± 1h day–night cycle in the propylene cages. The animals were sheltered for one week and prior to the experiment they were acclimatized to laboratory temperature. The protocol was approved by Animal Ethics Committee 1358/ac/10/CPCSEA constituted for the purpose as per CPCSEA Guideline

Acute Toxicity Studies:

Acute toxicity study was carried out by using graded doses seeds of Salvia hispanica and Linum usitatissium on albino rats. Seed powders were dissolved in 1% gum acacia and were administered intra-peritoneal in graded doses (200 to 2000mg/kg body weight). The animals were observed for 72 h for behavioural effects such as nervousness, ataxia, excitement, alertness, dullness and death. The LD50 was calculated as the geometric mean of the dose that caused 100% mortality and that which caused 0% mortality

Fourier Transform Infrared Spectrophotometer (FTIR)[13]

Fourier Transform Infrared Spectrophotometer (FTIR) is perhaps the most powerful tool for identifying the types of chemical bonds (functional groups) present in compounds. The wavelength of light absorbed is characteristic of the chemical bond as can be seen in the annotated spectrum. By interpreting the infrared absorption spectrum, the chemical bonds in a molecule can be determined. Dried powder of different solvent extracts of each plant materials were used for FTIR analysis. 10 mg of the dried extract powder was encapsulated in 100 mg of KBr pellet, in order to prepare translucent sample discs. The powdered sample of each plant specimen was loaded in FTIR spectroscope (Shimadzu, IR Affinity 1, Japan), with a Scan range from 400 to4000 cm 1 with a resolution of 4 cm.

Evaluation of in vivo Anti-Inflammatory Activity by Histamine induced hind paw oedema [14and15].

The anti-inflammatory activity of the seeds of chia and flax were determined using histamine induced rat paw oedema assay .Wister Albino rats of either sex weighing 150-250 grams were divided into seven groups of five animals each. The dosage of the drugs administered to the different groups was as follows.

Group-I- control (received only vehicle 1% gum acacia suspension)

Group II –Indomethacin (5 mg/kg,)

Group-III- 100 mg/kg b.w. Chia seeds

Group IV-200 mg/kg b.w Chia seeds

Group V- 100 mg/kg b.w. Flax Seeds

Group-VI -200 mg/kg b.w. Flax Seeds

Group-VII – 100 mg/kg Chia+ Flax seeds

Group VII-200 mg/kg Chia+ Flax seeds

All the drugs were administered orally. Indomethacin served as the reference standard anti-inflammatory drug. After one hour of the administration of the drugs, (histamine 1mg/ml) histamine solution in normal saline was injected into the sub plantar tissue of the left hind paw. The paw volume of the rats was measured in the digital plethysmograph, at the end of 0 min, 1 hrs, 2 hrs, 3 hrs. Mean increase in Paw volume and percentage of anti-inflammatory activity were calculated. The results were statistically analyzed by analysis of variance. Increase in paw thickness in control/ treatment

PC/PT = Pt – Po

Percentage of inhibition = PC – PT x 100 / PC

Where Pt = paw thickness at time t, Po = initial paw thickness, PC = Increase in paw thickness of control group and

PT = Increase paw thickness of the treatment groups

In vitro studies for determination of anti-inflammatory potential:

(A) The human red blood cell (HRBC) membrane stabilization method [16]:

The blood was collected from healthy human volunteer who had not taken any NSAIDS for 2 weeks prior to the experiment and mixed with equal volume of Alsever solution (2 % dextrose, 0.8 % sodium citrate, 0.5 % citric acid and 0.42 % NaCl) and centrifuged at 3,000 rpm. The packed cells were washed with isosaline and a 10 % suspension was made. Various concentrations of extracts were prepared (100 and 120 mg/ml) using distilled water and to each concentrations, 1 ml of phosphate buffer, 2 ml hypo saline and 0.5 ml of HRBC suspension were added. It was incubated at 37 0 C for 30 minutes and centrifuged at 3,000 rpm for 20 minutes and the hemoglobin content of the supernatant solution was estimated spectrophotometrically at 560 nm. Diclofenac (100 μg/ml) was used as reference standard and a control was prepared by omitting the extracts. The experiments were performed in triplicates and mean values of the three were considered. The percentage (%) of HRBC membrane stabilization or protection was calculated using the following formula:

Percent Protection (%) = (100- OD of drug treated sample/OD of Control) X 100.

(B) Inhibition of Albumen Denaturation [17]:

Method as prescribed was followed with minor modifications. The reaction mixture was consisting of test extracts and 1% aqueous solution of bovine albumin fraction, pH of the reaction mixture was adjusted using small amount at 37°C HCl. The sample extracts were incubated at 37°C for 20 minutes and then heated to 51°C for 20 minutes after cooling the samples the turbidity was measured spectrophotometrically at 660 nm. Diclofenac sodium was taken as a standard drug. The experiment was performed in triplicates. Percent inhibition of protein denaturation was calculated as follows:

Percent inhibition (%) = (OD of Control-OD of Sample/OD of Control) X100.

Statistical Analysis:

The mean value ± SEM was calculated for each parameter. The results were analyzed statistically by ANOVA is followed by Dunnet’s test. The minimum level of significant was fixed at p < 0.05

RESULTS AND DISCUSSION:

Phytochemical Screening:

Phytochemical screening of both chia and flax seeds showed that they contain flavonoids. The results are tabulated in table-2

Table 2: Results of phytochemical analyses of seeds of Salvia hispanica and Linum usitatissium

Test	*Salvia* *hispanica*	*Linum* *usitatissium*
Alkaloids	-	-
Glycosides	-	-
Proteins	+	+
Flavanoids	+	+
Carbohydrates	-	-
Steroids	-	+
Saponins	-	-

“+”- Indicates presence “_” indicates absence

FTIR Spectral Data Interpretation:

From the results obtained in the present study, it could be concluded that seeds of Salvia hispanica (Chia seeds) Linum usitatissium (Flax seeds) possess various functional groups indicate the presence of carbohydrates, carotenoid, glycogen, amino acids, amides, starch, calotropin, calotropogenin, phosphates, lipids, glycogen and cellulose. From the FTIR report it was analysed that there is no interaction between chia and flax seeds when given in combination. FTIR results were represented graphically and also tabulated in table-3 anfd graph 1-3 .

Table:3:FTIR spectral peak values and functional groups obtained for the seeds of Salvia hispanica and Linum usitatissium

Name of the plant

Peak values

Functional groups

SAMPLE-1:

Salvia hispanica(Chia seeds)

1162

1357

1442

1599

1627

1745

2918

2929

3347

C-O Group-I

C-H bending

C-H bendig

C=C group

C=Ocarbonyl group

C-H stretching

-OH group

SAMPLE-2:

Linum usitatissium (Flaxseeds)

1159

1354

1461

1599

1629

1745

2854

2929

3379

C-O Group-I

C-H bending

C-H bendig

C=C group

C=Ocarbonyl group

C-H stretching

-OH group

SAMPLE-3:

Salvia hispanica (Chia seeds) + Linum usitatissium (Flax seeds)

1161

1367

1447

1599

1627

1745

2864

2927

3372

C-O Group-I

C-H bending

C-H bendig

C=C group

C=Ocarbonyl group

C-H stretching

-OH group

Acute toxicity studies:

No mortality and behavioral changes were observed. Therefore, chia and flax seeds were safe up to 2000 mg/kg body weight dose. In accordance with this doses of 100, and 200 mg/kg body weight were selected for experimental studies .

Evaluation of In vivo anti -inflammatory activity:

The paw volumes and percentages of inhibition seeds of chia and flax and standard drugs are shown in it was observed that the seeds of chia and flax individually and combination showed significant inhibition at two doses. The inhibition was the highest at 3 h at 200 mg/kg dose which was slightly lower than Indomethacin effect. The results were tabulated in table-4 and also represented graphically in graph-4

Table: 4: Evaluation of in vivo Anti-Inflammatory Activity by Histamine induced hind paw oedema

Treatment	% Of Inhibition of Paw Oedema After
Treatment	1 hr	2 hr	3 hr
Group -I	-	-	-
Group -II	21	48.85 **	55.71 **
Group -III	19.45	28.42	32.14
Group -IV	26.93	31.64	36.19 *
Group -V	24.09	27.53	35.97 *
Group -VI	20.95	29.25 *	35.47 *
Group -VII	25.94	32.81 *	39.69 **
Group -VIII	27.85	36.36 *	41.2 **

* indicates p<0.05 ** indicates p<0.001 n=5

Evaluation of In vitro activity anti-inflammatory activity by the human red blood cell (HRBC) membrane stabilization method:

The phytochemical analysis of chia and flax showed that it is rich in flavanoids .In the study of membrane stabilization activity of chia and flax seeds at concentration range of 100-200 μg/ml protected significantly in a concentration dependent manner the erythrocyte membrane against lysis induced by hypotonic solution. Acetylsalicylic acid in the concentration of 100 μg/ml used as standard also offered significant (p<0.001) protection of RBC’s membrane against damaging effect induced by hypotonic solution.

Graph-4: Evaluation of in vivo Anti-Inflammatory Activity by Histamine induced hind paw oedema:

Table: 5 In vitro studies for determination of anti-inflammatory potential by the human red blood cell (HRBC) membrane stabilization method

Treatment	Concentration (μg/ml)	Absorbance at 660nm	% Inhibition of haemolysis
Control	-	0.045 + 0.98	--
Combination	100	0.021 + 3.78	53.3**
Combination	200	0.013 + 5.67	71.1**
Flax seed	100	0.031 + 1.34	31.1
Flax seeds	200	0.027 + 1.78	40.8*
Chia seeds	100	0.028 + 3.56	41.5*
Chia seeds	200	0.034 + 1.34	22.2
Diclofenac sodium	100	0.15 ± 0.01	51**

* indicates p<0.05 ** indicates p<0.001

Graph : 5 In vitro studies for determination of anti-inflammatory potential by the human red blood cell (HRBC) membrane stabilization method:

Table: 6 In vitro studies for determination of anti-inflammatory potential by protein denaturation

Treatment(s)	Concentration (μg/ml)	Absorbance at 660nm	% Inhibition of protein denaturation
Control		0.075 + 0.98	--
Combination	100	0.041 + 3.67	45**
Combination	200	0.033 + 1.45	56**
Flax seeds	100	0.051 + 3.56	32*
Flax seeds	200	0.047 + 6.78	45**
Chia seeds	100	0.058 + 3.67	37
Chia seeds	200	0.044 + 2.56	41*
Aspirin	100	0.22 ± 0.01	70**

* indicates p<0.05 ** indicates p<0.001

Graph-6: In vitro studies for determination of anti-inflammatory potential by protein denautration method:

CONCLUSION:

The result of our study showed a significant promising dose-dependent and synergistic reduction in inflammation by in vitro and in vivo methods.

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Phytoconstitutents	Test	observation
Tannins (Braymer’s Test	2ml extract + 2mlH2O+2-3drops FeCl3 (5%)	Green precipitate
Flavonoids	1ml extract + 1ml Pb(OAc)4(10%)	Yellow coloration
Terpenoids	2ml extract + 2ml (CH3CO)2O + 2-3 drops conc. H2SO4	Deep red coloration
Saponins (Foam Test)	a)5ml extract + 5ml H2O + heat b) 5ml extract + Olive oil (few drops)	Froth appears Emulsion forms
Steroids (Salkowski Test)	2ml extract + 2ml CHCl3 + 2ml H2SO4(conc.)	Reddish brown ring at the junction
Carbohydrates (Molisch’s Test)	2ml extract + 10ml H2O + 2 dropsEthanolic α-naphthol (20%) +2ml H2SO4(conc.)	Reddish violet ring at the junction
Glycosides (Liebermann’s Test)	2ml extract + 2ml CHCl3+ 2ml CH3COOH	Violet to Blue to Green coloration
Alkaloids (Hager’s Test)	2ml extract + few drops of Hager’s reagent	Yellow precipitate
Proteins (Xanthoproteic Test)	1ml extract + 1ml H2SO4(conc.)	White precipitate