In vivo investigation of the potential hypoglycemic activity of Pennisetum setaceum: Justification of the traditional use among Jordanians

 

Lidia K. Al-Halaseh1*, Nariman A. Al-Jawabri1, Hayat Al-Btoush1, Rawan Al-Suhaimat1,

Siba Majali1, Maha N. Abu Hajleh2, Ali Al-Samydai3, Mai A.M. Ahmed4

1Department of Pharmaceutical Chemistry, Faulty of Pharmacy, Mutah University,

Zip code (61710), Al-Karak, Jordan.

2Department of Cosmetic Science, Pharmacological and Diagnostic Research Centre,

Faculty of Allied Medical Science, Al-Ahliyya Amman University, Zip code (19328), Amman, Jordan.

3Pharmacological and Diagnostic Research Centre, Faculty of Pharmacy,

Al-Ahliyya Amman University, Zip code (19328), Amman, Jordan.

4Department of Pharmacognosy, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt.

*Corresponding Author E-mail: drhalaseh@mutah.edu.jo, suh-rawan@mutah.edu.jo

 

ABSTRACT:

Objectives: Pennisetum setaceum has been used traditionally as herbal hypoglycemic and slimming medicine by Jordanians. However, its claimed benefits are still elusive. The current in vivo study aimed to justify the folk use of this herbal tea and evaluate its effect. Materials and methods: plant material was collected from South Jordan and identified. Crude aqueous extract of Pennisetum setaceum aerial parts was prepared. The lethal dose was estimated after given groups of mice oral doses of the herbal extract (1, 2, 4, 6, 8 and 10 g/Kg mouse weight). The potential weight reduction activity was measured by recording the animal weight for 30 days after feeding them 1 g/Kg oral doses of the herbal medicine for 10 consecutive days. The hypoglycemic activity was evaluated in two different concentrations: 1 and 2 g/Kg mouse weight. Male mice with an average weight of 26.5 ± 2.76 g were rendered diabetic by an intraperitoneal dose of streptozocin. Groups of mice were given the herbal medication orally. A negative control group was given d. water. Blood sugar levels were measured from tail blood every 30 min for 3 h. LD50 was estimated. Results: The plant was identified to be P. setaceum by the local authority in Jordan. LD50 was estimated to be 10 g/Kg mouse weight after oral administration. Significant weight reduction of the orally fed group (27 ± 3.1 to 24 ± 2.2) with minor undesirable effects. A significant decrease in blood glucose level in the treated diabetic animals were measured compared with the control group. Significant variation in time-dependent hypoglycemic effect was found between the two tested doses. Conclusion: P. setaceum aqueous extract can be considered as a promising candidate for amelioration of blood sugar profile with potential slimming activity.

 

KEYWORDS: Hypoglycemic, Medicinal plants, Pennisetum setaceum, Fountain grass, Slimming, Diabetes, Natural products.

 

 


INTRODUCTION:

Despite the lack of medical evidence and scientific knowledge, Jordanian have a strong belief in herbal medication. Their use is not limited to minor health conditions but for treating some major concerns include hyperglycemia, dyslipidemia and cancer.1,2

 

Although they have experienced side effects, natural remedies for weight reduction were used by 23.6% among those who resort to herbal medications in Jordan according to a recent report published by Abdel-Qader and his colleagues.3

 

Sadly, the prevalence of diabetes among Jordanians is high and on an increasing trend.4 The case in Jordan is a reflection of the whole world’s scenario. It is estimated by the WHO that the total diabetic cases will reach 266 million by 2030.5 Obesity prevalence in Jordan is also alarming; the ubiquity of obesity for men was 28.1%, while the percentage almost doubled for women with 53.1%.6  Treatment classified under the categories of appetite suppressants, metabolic promotors, digestion and absorption blockers, and the most recent; obesity gene product inhibitors.7 Unfortunately, none of them has good efficiency with unremarkable side effects. Obesity is considered a predisposing factor in diabetic patients.8,9

 

The scenario in Jordan mirrors the behaviour of people in the Middle East as well. Individuals are seeking the help of Medicinal plants in treating their illnesses. The traditional recipes are available and hereditary for a wide range of diseases and symptoms starting from mild cases of diarrhoea to even more complicated illness including cancer. Recently, herbal medications were also tested for their activity against COVID-19 pandemic.10-12

 

Although community pharmacies stocked conventional medicines for aiding weight loss and regulating blood sugar, most patients prefer using herbal remedies despite the lack of clinical evidence. 13-16 Extra health benefits were expected by users of natural weight reduction remedies include improving the lipid profile and keeping blood glucose into normal range such as with cinnamon, flaxseed, ginger, Nigella sativa, green tea, nut grass and many others.17-23 Other illnesses were also reported to be treated by natural remedies, for instance, ocular problems, hepatotoxicity, and anaemia.24,25

 

Unfortunately, people seek an easy and fast way to decrease blood sugar levels that’s mostly because of the misconception of the absolute safety of natural drugs. The unknown active ingredients and the absence of a clear explanation of the mechanism of action make the harmful side effects unpredictable. Herbal mixtures abused for claimed health beneficence, include the potent Ephedra sinica and anthraquinones laxative, even though they have a well-known hepatic- and nephrotic toxicity.26,27

 

Pennisetum setaceum, fountain grass, or halfa-bar as called by locals in Jordan, is not among the commonly used medicinal plant with known biological and therapeutic uses. P. setaceum is a grass that belongs to Poaceae family. Globally, it is recorded as native plant to Jordan where, at the national level, it is classified as engendered species according to the report released by the Jordanian Royal Society for the conservation of nature (RCSN). This species is distributed in the Mediterranean ecozone and could be cultivated in another region. Mainly it is used as an ornamental plant and sometimes used for its medicinal characteristics by local communities.28,29 Recently, it was reported that a clinical reduction in both the blood sugar level and body mass index in an overweight woman after self-prescribed the plant extract. 30

 

The anthocyanin-rich aqueous extract of P. setaceum might be responsible for its biological activity.31 The plant was also studied to be a ruminant feed in areas suffering from forages shortage.32 Unfortunately, no data is available, up to our knowledge, regarding the safety profile of the plant.

 

MATERIALS AND METHODS:

Plant material:

Dried leaves of P. setaceum were collected from areas in Al-Karak, south Jordan, in October 2020. The leaves were chopped into small pieces using a kitchen appliance. The plant is known as Halfa-Bar by locals in Al-Karak. Identification and authentication of the plant were done by experts at the Royal Society for the Conservation of Nature (RSCN), Amman, Jordan. A voucher sample was deposited in the RSCN herbarium.

 

Preparation of the extract:

Each 250 g of reduced-size and dried leaves was macerated with 100 ml distilled water with continuous stirring using mmemert shaker waterbath® at 25℃ for 16 h. The suspension was filtered twice, then concentrated until a fine powder was obtained using Benchtop Manifold Freeze Dryer from Millrock Technology®, UK.

 

Experimental animals:

Male albino mice, the average weight of 26.5 ± 2.76 g, were obtained from the animal house, faculty of pharmacy, Mutah University. Mice housing, food and environment are following the ethical guidelines for animal protection in the faculty. The mice were moved from the animal house to the animal laboratory, a week before the experiment, to be acclimatized.

 

LD50 measurement:

Acute toxicity and lethal dose determinations were carried out to establish the therapeutic index of P. setaceum aqueous crude extract. The experimental animals were distributed randomly into four groups of 6 mice each. The animals have no access to food 6 h before the experiments. Oral doses of a final volume of 50 μl were given using an oral gavage apparatus. The tested doses were: 1, 2, 4, 6, 8 and 10 g/Kg separately. The Control group was treated with distilled water only. The experimental animals were monitored for mortality 24 h after treatment and 14 days after the administration of the prepared extract. The lethal dose was estimated depending on the mortality rate of 50% of the tested animal in a group after 24 h. General health status was observed for 30 days after the treatment. 

 

Weight reduction studies:

10 male mice with an average weight of 27 ± 3.1 were fed with daily oral doses of 1 g/Kg P. setaceum aqueous extract for 10 days in addition to the standard ad libitum. The plant extract was suspended in 200 μl and delivered to the animal via intragastric route using oral gavage intubation apparatus. Average weight was measured 10 days, 20 days, and 30 days later. The general health status of the treated animals was observed.

 

Streptozocin STZ-induced hyperglycemia:

The experimental animals were deprived of food and water 4 h before in vivo treatment. Mice were rendered diabetics by an intraperitoneal high single dose of streptozocin (STZ) in citrate buffer (200 mg/Kg, pH 4.5) prepared following published reports.33 The tail end was cut from under strained mice and blood drops from a cut tail end were used to determine FBS using the Joycoo® glucose meter. Fasting blood sugar (FBS) was measured the following day. Values of FSB range of 275-400 mg/dl is considered diabetic.34 After being given the STZ and measuring FBS, the mice have free access to food and water.

 

Administration of the P. setaceum aqueous extract:

For the administration of the herbal medication, two groups of 12 mice were given 2 and 1 g/Kg of the extract separately. The medication was delivered orally and slowly using intragastric intubation apparatus. The experimental animals were deprived of food 16 h before oral medication was given. Every dose of the freeze-dried plants was suspended in 200 μl distilled water. FBS was measured for each treated diabetic mouse at zero time, then every 30 min after the oral dose for 3 h.35 Negative control groups (diabetic mice) were given distilled water. The positive control group (diabetic mice) was given short-acting insulin NovoMix® 30 (10 μl, 1 IU) and then blood sugar levels were recorded for 30 min in a time interval of 5 min. Norm-glycemic group (no induction of diabetes) was given the plant aq. Extract 1 and 2 g/Kg to evaluate the hypoglycemic effect. No anesthesia was needed either during medication or during blood sampling.

 

RESULTS AND DISCUSSIONS:

Plants have a significant contribution to medicine but, their self-prescription need better organization based on clinical evidence. Although P. setaceum is not a popular medicinal plant in Jordan, it is traded as a slimming agent between folk in south Jordan. Lack of studies linking the anti-diabetics and weight-reduction potential of the medicinal plants urges the need to measure the hypoglycemic activities of those agents used traditionally for slimming purposes.36

 

LD50 and Toxicity observations:

The recorded value of LD50 was 10 g/Kg of animal weight. Toxicity signs of a bloated belly, reduced appetite, decreased activity, mood changes were observed also. High lethal doses indicate the safety of the plant when given orally.

 

Weight reduction studies:

No significant decrease in weight was noticed after 10 days. While the average weight was significantly reduced in the second and third part of the month from 27 ± 3.1 to reach 24 ± 2.2 g (p ˂ 0.005) at the end of the experiment. 

 

Hypoglycemic activity:

48-h after STZ injections, blood glucose measurements were 438.44 ±101 mg/dl. The mice were confirmed to be diabetics.34

 

A significant decrease in blood glucose levels in treated experimental animals was measured compared to the control group. Significant variation in time-dependent hypoglycemic effect in the treated groups were computed. Those diabetic animals that have been treated with insulin, showed a significant rapid drop in the glucose level just in 5 min after the i.p dose. Blood glucose readings sharply declined to reach 70 ± 24 mg/dl after 30 min of a single dose (1IU) of insulin.

 

Statistics were conducted using SPSS software; Statistical Package for the Social Sciences® software, version 21. Values were considered significantly different if P value ≤ 0.05. Ratios of glycemic reduction in the groups treated with 1 and 2 g/Kg of the extract compared to the negative controlled group (treated with d. water) were shown in Table 1.


 

 

Table 1: Ratio of glycemic reduction

Descriptive

 

 

Multiple Comparisons

Groups

N

Mean

Std. Deviation

Minimum

Maximum

F

Sig

(I) Group

(J) Group

Sig.

2g/Kg

6

34.9395

16.01125

9.87

53.73

7.448

0.006

2g/Kg

1g/Kg

0.562

1g/Kg

6

29.9734

19.09621

6.89

51.82

2g/Kg

d.water

0.003

d.water

6

4.8216

3.01654

1.59

10.02

1g/Kg

d.water

0.009

 


Table 1 clearly showed that the average ratio of glycemic reduction was found to be different across groups, F (2, 15) = 7.448, p = 0.006. The LSD multiple comparisons performed at the 0.05 significance level found that the mean average ratio of glycemic reduction in the control group that was treated with d. water (M = 4.8216, SD = 3.01654, N = 6) was significantly lower than that for the diabetic group treated with 1g/Kg of the herbal medication (M = 29.9734, SD = 19.09621, N = 6) and 2 g/Kg of the herbal medication (M = 34.9395, SD = 16.01125, N = 6) with p-value; 0.009 and 0.003 respectively. On the other hand, data conclude that there are no significant differences between the average ratio of glycemic reduction readings in the treated groups (1 g/Kg and 2 g/Kg) with a p-value of 0.562. Data obtained from the treated groups with the extract showed a clear pattern of time-depended anti-hyperglycemic activity in diabetic animals compared to the negative control group (treated with distilled water) as shown in figures 1 and 2.

 

Figure 1: Ratio of blood glucose level reduction between the tested groups

 

Figure 2: Ratio of blood glucose level reduction between the tested groups in time scale

 

As shown in Table 2, the Model was estimated to investigate whether time and group could predict the changes in the average ratio of glycemic reduction across together. The predictors accounted for a significant amount of variance in the average ratio of glycemic reduction. Overall, the model accounted for approximately 75.9% of the variance in the outcome. Adjusted R Squared = 0.759 with a significant relationship (p < 0.001). The model used helps to explain 78.7% variation in the dependent variable. Both of the treated groups (2 and 1 g/Kg) showed stronger relationships between dependent and independent variables with R = 0.986 and 0.984 respectively in comparison with d. water (R=0.887). The outcome of investigating the relationship between dose and time is shown in table 2.

 

Table 2. Study estimating the relationships between dose and time

Model

R

R Square

Adjusted R Square

Std. Error of the Estimate

2 g/Kg

0.986a

0.972

0.965

2.97792

1 g/Kg

0.984a

0.969

0.961

3.74962

d. water

0.642a

0.412

0.265

2.58657

Overall model

0.887a

0.787

0.759

9.43365

a. Predictors: (Constant), VAR00002

 

CONCLUSION:

In conclusion, the observed and measured data confirm, with experimental evidence, that P. setaceum extract participates in weight reduction, therefore, enhancing the obesity profile if conjugated with a healthy lifestyle. Moreover, the research output confirms the hypoglycemic activity of the tested medicinal plant in vivo. Time-dependent variation was measured between the tested, low and high, therapeutic doses. The plant extract has the potential to decrease blood glucose levels accordingly, to treat diabetes. Further investigations for the safety of its use as slimming and hypoglycemic agents are essential. Exploring the hypoglycemic mechanism of action of the plant extract was also recommended.

 

ACKNOWLEDGMENTS:

The authors would like to acknowledge the support of Mutah University, Faculty of Pharmacy, for the laboratories facilities given during this research work. The authors acknowledge the Royal Society for the conservation of Nature (RCSN), Amman, Jordan for plant identification and taxonomy work.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

DATA AVAILABILITY STATEMENT:

The datasets generated during and/ or analysed during the current study are available from the corresponding author on reasonable request.

 

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Received on 29.12.2021           Modified on 07.04.2022

Accepted on 15.06.2022         © RJPT All right reserved

Research J. Pharm. and Tech. 2022; 15(7):3185-3189.

DOI: 10.52711/0974-360X.2022.00533