Hypolipidemic Activity of Combination of Extract of Zingiber officinale Rosc. (Ginger) Rhizomes and Citrus aurantifolia Linn. (Lime) Fruits Juice on Triton-Induced Hyperlipidemic Rats

Khine Khine Lwin, Mu Mu Sein Myint, Khin Phyu Phyu1, Aye Win Oo1, Phyu Phyu Win1, Khin Tar Yar Myint1, Myint Myint Khine, Hlaing Myat Thu, Kyaw Zin Thant

Myanmar Health Sciences Research Journal, 2020; 32(2): 173-179

This study was done in 2016-2017to investigate the hypolipidemic activity of combination of watery extract of ZingiberofficinaleRosc.(ginger) rhizomes and fruit juice of Citrus aurantifolia Linn. (lime) in triton-induced hyperlipidemic rats. Acute oral toxicity test of combination of watery extract of ginger and lime juice was done on albino mice by the method of Litchfield and Wilcoxan (1949). Phytochemical tests of the combination of ginger extract and lime juice were also performed. Twenty-four Wistar albino rats of both sexes were randomly divided into four groups consisting six rats each. Group 1 was normal control group. Group 2 served as triton-induced hyperlipidemic control group. Group 3 was given combination of watery extract of ginger and lime juice (10g/kg) and Group 4 was given standard drug (atorvastatin 30mg/kg). Fasting serum lipid levels (total cholesterol, high density lipoprotein (HDL) and triglyceride) of all rats were determined with Semi Auto Chemistry Analyzer. Low density lipoprotein (LDL) levels of the rats were calculated by using formula. It was observed that LD50 value of the combination of ginger and lime was more than
16 g/kg. The results showed that after two weeks of pretreatment, serum total cholesterol and LDL levels were reduced significantly in the combination of ginger extract and lime juice (10 g/kg) treated group (p<0.001) when compared with hyperlipidemic control group by using unpaired Student`s t test. In conclusion, the combination of watery extract of ginger and lime juice possessed significant hypolipidemic effect on serum total cholesterol and LDL levels in triton-induced hyperlipidemic rats.

Yield percentage of watery extract of fresh rhizomes of Zingiber officinale Rosc. was 4g per 100 g (4 %).

Acute toxicity test of combination of watery extract of Zingiber officinale Rosc. (ginger) rhizomes and juice of Citrus aurantifolia.(lime) fruits

It was found that the combination of watery extract of ginger rhizomes and juice of lime fruits showed no toxic effect and lethality in mice upto the maximum dose level of 16 g/kg bwt. Therefore, medium lethal dose (LD50) of the combination was found to be more than 16 g/kg.Histopathological examinations of the organs of the mice were not done.

Phytochemical analysis of combination of watery extract of Zingiber officinale Rosc. (ginger) rhizomes and juice of Citrus aurantifolia. (lime) fruits

The results are shown in Table 1.

Table 1.Phytochemical analysis of combination of watery extract of Zingiber officinale Rosc. (ginger) rhizomes and juice of Citrus aurantifolia (lime) fruits

(+)=presence, (-)=Absence

Effect of combination of watery extract of Zingiber officinale Rosc. (ginger) rhizomes and juice of Citrus aurantifolia. (lime) fruits on serum lipid profile level of triton-induced hyperlipidemic rats

The results are shown in Table 2.

Table 2. Effect of combination of watery extract of ginger rhizomes and juice of lime fruits on serum lipid profile level of triton-induced hyperlipidemic rats

Results are shown in mean±SD
*=p<0.05,**=p<0.01,***=p<0.001

Statistical comparison was made between the control and triton-induced hyperlipidemic control group.Statistical comparison was made between the triton-induced hyperlipidemic control group and combination of watery extract of ginger rhizomes and juice of lime fruits, as well as atorvastatin receiving groups.

Mean serum total cholesterol, low density lipoprotein (LDL), triglyceride and HDL levels of the control group (distilled water only receiving group) were 105.67±3.67 mg/dl, 27.5±3.39 mg/dl, 102.17±20.06mg/dl and 57.33±7.84 mg/dl, respectively.Mean serum total cholesterol, LDL, triglyceride and HDL levels of triton-induced hyperlipidemic control group were 363.63±41.46 mg/dl, 199.55±37.09 mg/dl, 6438 mg/dl and 97.57±14.04 mg/dl, respectively. Significant increases in serum total cholesterol, LDL, triglyceride, HDL levels were found when compared with those of the control group (distilled water receiving group) (p<0.001).

Mean serum total cholesterol, LDL, trigly-ceride and HDL levels of triton-induced hyperlipidemic group administered with combination of watery extract of ginger rhizomes and juice of lime fruits,at the dose of 10 g/kg were 254.84±24.31 mg/dl, 92.33±38.13mg/dl, 6026 mg/dl and 131.74±54.01 mg/dl, respectively. There were significant decreases in serum total cholesterol (TC) and LDL levels (p<0.001) except triglyceride level when compared with the hyperlipidemic group. Reduction of triglyceride level was 6.4%. So, there was no significant reduction in serum triglyceride level. Mean serum HDL level was increased but it was not significant.

Mean serum total cholesterol, LDL, trigly-ceride and HDL levels of triton-induced hyperlipidemic group administered with standard drug atorvastatin (30 mg/kg) were 375.04±97.99 mg/dl, 175.67±68.54mg/dl, 6438 mg/dl and 140.82±42.08 mg/dl, respectively. There was no significant decrease in serum total cholesterol and LDL levels and triglyceride level. But significant increase in mean serum HDL level was found (p<0.05). Statistical comparison was made between the control and triton-induced hyperlipidemic control group.Statistical comparison was made between the triton-induced hyper-lipidemic control group and combination of watery extract of ginger rhizomes and juice of lime fruits, as well as atorvastatin receiving groups.

Table 3.   Percent reduction of mean total cholesterol,LDL and triglyceride levels

TC=Total cholesterol, LDL=Low density lipoprotein

Percent reductions of mean serum total cholesterol level and LDL level with combination of watery extract of ginger rhizomes and juice of lime fruits  were 29.92% and 53.73%,respectively, as shown in Table 3.

There are many medicinal plants such as Phyllanthus emblica Linn.,Zingiber officinale,Allium sativum Linn. etc which have been used throughout the world for the treatment of hyperlipidemia.4 Zingiber officinale Rosc. (Family-Zingiberaceae) commonly known as ginger is native to Asia such as China, Japan and India.It grows and is also cultivated widely throughout Myanmar. Rhizome of ginger is used traditionally for sore throat, fever, cough, tooth ache, joint pain and inflammation, vomiting and indigestion in Myanmar and India.4, 5It was reported that Zingiber officinale Rosc. rhizome has anti-inflammatory,6, 7 hypoglycemic,8antioxidant,5 hypo-lipidemic5 and antipyretic activities.5 Ginger extract showed hypolipidemic effect in hypercholesterolemic rabbits.9

Citrus aurantifolia Linn. (lime) (Family-Rutaceae) fruit is Than-payar in Myanmar. It grows and also cultivated widely throughout Myanmar. In traditional medicine of Myanmar and India, fruits of Citrus aurantifolia Linn.are useful in cough, sore throat, fever, joint pain, bronchitis and cough. The fruit is used as antiseptic.4Kumariet al. reported that juice of fruits possessed strong antioxidant activity to provide protection against damage to biomolecules by using DPPH assay method.10

It was reported that extract of Citrus aurantifolia Linn. fruits had antimicrobial activities.11, 12 BoshtanMaryanet al (2011) reported that Citrus aurantifolia Linn. had antioxidant activity, in vitro antibacterial activity and hypocholesterolaemic activity.13

There is no scientific report of hypo-lipidemic effect ofcombination of extract of Zingiber officinale Rosc. rhizomes (ginger) and Citrus aurantifolia fruits (lime) juice in Myanmar. This study was performed to investigate the hypolipidemic activity of combination of watery extract of Zingiber officinale Rosc. rhizomes and Citrus aurantifolia fruits juice on triton-induced hyperlipidemia in albino rats. Acute toxicity study of the combination was also performed in albino mice.

It was a laboratory-based experimental animal study.

Study area

Study area was Pharmacology Research Division, Department of Medical Research.

Plant materials and preparation

Zingiber officinale Rosc.rhizomes (ginger) and Citrus aurantifolia. fruits (lime) were purchased from Thirimingalar market in Yangon and identified by the Botanist at Pharmacology Research Division, Department of Medical Research. Fresh ginger rhizomes were washed with water and cut into small pieces. Then, 100 g of rhizomes were extracted with 1 litre of distilled water in boiling water bath for 6 hours to get watery extract.The extract solutions were filtered and the filterates were dried in 100°C water bath to get dried extract. The watery extracts obtained were stored in airtight desiccator for further use in experiment.The fresh juice of Citrus aurantifolia fruits (lime) was obtained by squeezing. Combination of watery extract of Zingiber officinale Rosc.rhizomes (5g/kg) and freshly prepared juice of Citrus aurantifolia. fruits (5g/kg) were used in this study (Wt/Wt) (1:1 combination) (i.e 10 g/kg). The combination was dissolved in distilled water before use in the animal experiment. This ratio of combination was extrapolated from human use.

Phytochemical analysis

Phytochemical tests of combination of watery extract of Zingiber officinale Rosc. rhizomes and juice of Citrus aurantifolia fruit were performed qualitatively by using the method of Harborne.14

Acute toxicity study

Albino mice (ddy strain mice) of both sexes weighing between 25g-30g were used for acute toxicity test of combination of watery extract of Zingiber officinale Rosc. rhizomes and juice of Citrus aurantifolia fruit. Acute toxicity test were performed and calculation of LD50 value was done according to the method of Litchfield and Wilcoxan (1949).15 The doses used in this study were 2.5 g/kg, 5 g/kg, 10 g/kg and 16 g/kg bwt. They were observed for toxic signs and mortality daily for 14 days.

Determination of hypolipidemic activity of combination of watery extract of Zingiber officinale Rosc.rhizomes and juice of Citrus aurantifolia. fruit ontriton-induced hyperlipidemic rats

Adult healthy albino rats (Wistar strain) of both sexes weighing (200g-250g) obtained from Animal Service Division (Department of Medical Research) were used in this experiment. In the pilot study, after the mice were kept fasting for 18 hours, intraperi-toneal injections of Triton WR 1339, 300 mg/kg in 0.9% physiological saline solution to 6 rats were done to induce hyperlipidemia. After 24 hours of triton injection, under chloroform anesthesia, blood samples were collected by cardiac puncture to determine fasting serum lipid profile. Blood samples were collected after 18 hours of fasting.

It was found that there were no increases in serum lipid levels of the rats from baseline levels. Therefore, intraperitoneal injections of triton 400 mg/kg in 0.9% physiological saline solution to the rats were chosen to induce hyperlipidemia for this study. In literatures, intraperitoneal injection of doses of triton ranged from 250 mg/kg to 400 mg/kg in 0.9% physiological saline solution to the rats was used to induce hyperlipidemia for screening of hypolipidemic activity of the new drugs in rats.16, 17, 18

In this study, twenty-four albino rats of both sexes were divided into six groups and each group contained 6 animals. Group 1 was given distilled water only (negative control group). Group 2 was hyperlipidemic control group which did not receive any treatment. Group 3 was given orally combination of watery extract of Zingiber officinale Rosc.(ginger) rhizomes (5g/kg) and juice of  Citrus aurantifolia. (lime) fruits (5 g/kg) (i.e the combination of test drug 10 g/kg) (1:1 combination Wt/Wt). This combination was dissolved in distilled water before oral administration to animals. Group 4 was orally given standard drug atorvastatin 30 mg/kg and served as a standard drug group.

Combination of watery extract of ginger rhizomes and juice of lime fruits, atorvastatin and distilled water were administered orally once daily to the rats for 14 days. On day 14 of treatment, after the animals were kept fasting for 18 hours, Group 2 to group 4 were given single dose injection of triton 400 mg/kg in 0.9% physiological saline solution intraperitoneally to induce hyperlipidemia.19-21

On day 15 (i.e 24 hours after administration of triton injection) after 18 hours of fasting, under chloroform anesthesia, blood samples were collected by cardiac puncture to determine fasting serum lipid profile. In this study, fourteen days of pretreatment with the test drug before intraperitoneal injection of triton to the rats was done to get good therapeutic effect.Fasting serum lipid levels (i.etotal cholesterol, triglyceride, high density lipoprotein) of each rat from all groups were determined by using commercial kits (Hospitex Diagnostics, Italy) method and Semi Auto Chemistry analyzer. Low density lipoprotein level was calculated by using Cordova and Cordova formula (2013).22

Cordova and Cordova formula for calculation of LDL level was:

Data analysis

The results were shown in mean±SD. Unpaired Student t test was used to observe the significance of difference between means of the control and experimental groups. Values with p<0.05 was considered as statistically significant.

Cardiovascular diseases including atherosclerosis are the most common cause of mortality and morbidity worldwide. Hyperlipidemia contributes significantly to the manifestation and development of atherosclerosis and heart disease.1, 2Various plants were shown to be helpful in lowering plasma cholesterol and hyperlipidemia. Ginger rhizomes and lime juice had been reported to have antioxidant and hypolipidemic activities.5, 23 Oral ingestion of ethanol extract of ginger has been shown to have hypolipidemic and hypocholesterolemic effects in cholesterol fed rabbits.9In this study, hyperlipidemia was induced by intraperitoneal injection of Triton WR 1339.Rodolfo Paoletti and Remo Fumagalli described that Triton WR 1339 is one of the many well known nonionic detergents (surfactant) that induces the elevation of plasma cholesterol and triglyceride levels by increasing the hepatic cholesterol synthesis. The sustained hyperlipaemia and hypercholesterolaemia over 7 days are induced by interference with the uptake of plasma lipids by tissues. As a reaction to the impaired uptake of plasma cholesterol, the rate of endogenous cholesterol biosynthesis is rapidly increased in the liver.20

The administration of the surface active agent, triton WR 1339 induces hypercholesterolaemia and hyperlipaemia in many species. Triton is used to induce hyperlipidemia in rats because it has rapid onset of action and causes marked hyperlipidemia. The triton-induced acute hyperlipidemia in rat model is highly used in evaluation of the newer antihyperlipidemic drugs. Triton-induced hyperlipidemia in rats is simple and rapid for evaluation of test substance and can be considered as useful method for screening of activity of new antihyperlipidemic drugs.24In this acute toxicity study, the combination of watery extract of ginger rhizomes and juice of lime fruits showed no toxic effect and lethality in mice up to the maximum dose level of 16g/kg. Therefore, LD50 value was found to be more than 16g/kg. The results indicated that the combination of watery extract of ginger rhizomes and juice of lime fruits was found to be nontoxic.In this study, there were marked increases in the levels of serum total cholesterol(TC), triglyceride(TG), low density lipoprotein (LDL) and increase in the level of good cholesterol (HDL) in the rats administered intraperitoneally with triton (400mg/kg)(p<0.001).In our previous study of hypolipidemic activity, the combination of juice of ginger rhizome (2 g/kg) and lime fruit juice (2 g/kg) (Wt/Wt) (i.e the combination of the test drug, 4g/kg) was given orally to the rats for two weeks.

Then, the rats were intraperitoneally injected with Triton WR 1339 (400 mg/kg) to induce hyperlipidemia. Twenty-four hours after triton injection, thefasting serum lipid levels were determined. The results showed that there were no significant reductions in serum lipid levels in the rats.25In the present study for determination of hypolipdemic activity in rats, oral administration of the combination of watery extract of ginger rhizomes (5g/kg) and juice of lime fruit (5 g/kg) (i.e the combination of test drug 10g/kg) was used. It was observed that combination of watery extract of ginger rhizomes and juice of lime fruits at the dose of 10g/kg decreased the levels of total cholesterol (TC) and low density lipoprotein (LDL) cholesterol significantly but serum trigly-ceride (TG) levels were not decreased when compared with hyperlipidemic control group.

Mean serum HDL level was not significantly increased. In the present study, standard drug atorvastatin (30 mg/kg) showed significant increase in HDL level but there was no significant decrease in serum TC, TG, LDL levels when compared with hyperlipidemia control group.Plant constituents such as saponin, polyphenol, flavonoids and steroids have been reported to have hypolipidemic activity.26In the present study, the hypolipidemiceffectcan be due to the presence of polyphenols, flavonoids, steroids and saponin in combination of watery extract of ginger rhizomes and juice of lime fruits.

Conclusion

Combination of watery extract of Zingiber officinale Rosc.rhizomes and juice of Citrus aurantifolia fruits had significant lipid lowering effect (hypolipidemic effect) on serum total cholesterol and LDL levels in triton-induced hyperlipidemic rats.

The authors declare that they have no competing interests.