INTRODUCTION:

Medicinal plants are very precious to mankind in order for them to live a healthy and long life. Emblica officinalis belonging to the family Euphorbeaceae is one of such plant used in Ayurveda and for various indications and to increase the vigor and restore the lost energy. It is also called as “Indian gooseberry” or “Amla” and has been popularized for rejuvenation in Ayurvedic medicine since ancient times¹. Fruits of Amla contain a high concentration of polyphenols (gallic acid, ellagic acid etc), as well as amino acids, multiple vitamins, minerals, fixed oils, and flavonoids (rutin, quercetin etc)^2-4. The extracts obtained from various parts of this plant have been reported for the management of plenty of illness, including hyperlipidemia, inflammation, osteoporosis, neurological diseases, cancer, high blood pressure, and lifestyle diseases, as well as parasitic infections¹.

It also has antipyretic, analgesic, antimicrobial, antitussive, chemopreventive, cardioprotective, immunostimulatory, hepatoprotective, radioprotective, and hemoglobin-increasing properties⁵. The antioxidant constituents present in amla were found to be responsible for the regulation of various biochemical pathways and prevention of cell death.

Analysis of active components in herbal extracts or formulations is very important. An accurate fingerprinting method for analysis of amla in Amlakyadi Churna and an ultra-fast liquid chromatographic method for simultaneous estimation of ascorbic and gallic acid has been developed by various researchers^{6, 7}. In one study. nutrient medium containing Amla as a solid substrate has been used for the production of Naringinase enzyme⁸. Plenty of polyherbal formulations like herbal toothpaste, floating tablets, hair oil etc have been developed in the past two decades and contains amla as one of the principal ingredients^9-11. Amla extract and its formulation with other herbal ingredients have been reported to be beneficial in the management of uterine fibroids, gastroesophageal reflux disease, anti-aging and iron deficient anemia^12-15.

The benefits of amla for the management of lipid profile, especially total cholesterol (TC) and triglycerides (TG) are among the herb's key characteristics. In clinical research involving humans and experimental animals, the extract has been proven to effectively treat dyslipidemia without major side effects^{16, 17}. The flavonoids from Emblica officinalis have been proven in vitro to successfully lower cholesterol levels in blood serum and tissues¹⁸. Acute oral administration of methanolic extract of Emblica officinalis fruit, LD₅₀ was found as 1125mg/kg in Wistar rats⁵. Another report on toxicity evaluation detailed LD50 greater than 5000 mg/kg in rats for a water extract from Amla fruits¹⁹. These authors also reported the safety of aqueous extract at 1200mg/kg daily for a period of about 9 months in rats.

The extracts used in all of the aforementioned research on the toxicity of amla came from the pulp or juice of the fruit. The amla extract from the whole fruit, however, has been deemed superior because of the numerous polyphenols, triterpenoids, and fatty acids that it contains. We have developed an amla extract from whole fruit and standardized with alpha-linolenic acid (ALA) and alpha linolenic acid enriched diacyl-glycerol (ALA-DAG). In a double blind, placebo controlled, multicenter clinical study on 98 dyslipidemic patients, the whole fruit amla extract (Tri-Low^®; standardized to contain not less than 5% ALA) has exhibited reduction in TC and TG numbers along with ratio of lipids, atherogenic index of plasma and apoB/apo A-I ratio²⁰. In this study, decrease in cholesterol was observed without any deficiency in CoQ10 values. The authors concluded that the amla extract from whole fruit can be used for the management of lipid profile in general and dyslipidemia coupled with diabetes.

The effects of ALA on visceral fat are strengthened by the diacylglycerol (DAG). In a 90 days study on humans, it was found that adding ALA-DAG to a regular diet decreased body mass index, visceral fat and serum triacyl glycerol (TAG) in overweight men and women²¹. The postprandial fat oxidation in healthy adults was increased by ALA-DAG in another human study²². The investigators came to the conclusion that frequent ingestion of ALA-DAG improves metabolism of fat after the intake of fat rich meal, which can help to define some of its ability to reduce the area of visceral fat. When compared to TAG consumption, ALA-DAG intake considerably improved utilization of ingested fat as energy²³. Additionally, ALA-DAG has been shown to lower postprandial serum triglyceride (TG) levels in people who have normal or slightly elevated fasting TG levels²⁴.

This is the first time that amla extract has been standardized using ALA and ALA-DAG. Since amla extract contains these omega 3 fatty acids, which are highly useful for general metabolic health, it is crucial to assess the extract’s safety in accordance with industry standards. As per the standard regulatory guidelines, the goal of the current study was to assess the toxicity and safety of full spectrum amla fruit extract (Tri-Low^®) in rats.

MATERIALS AND METHODS:

Chemicals and test system:

Standardized amla extract (Tri-Low®) from Arjuna Natural Private Ltd, Kerala, India was the test product in this study. The Tri-Low®was standardized with total polyphenol content not less than (NLT) 35%, triterpenoids NLT 7%, Omega 3 fatty acids (total ALA) NLT 5% and ALA-DAG NLT 4%.

In-house bred Sprague Dawley rats were used for the safety studies. At the start of the study, animals were 8-10 weeks old and acclimatized to the animal house conditions (Temperature: 24±2°C; Humidity 50-70 %; 12/12-hour light/dark cycle) for one week. Potable drinking water and standard rat diet was given ad libitum. The protocol for this study was duly approved by the Institutional Animal Ethics Committee of Arjuna Natural Private Ltd., Kerala with approval number ANEL/IAEC/2016-II/1612019.

Acute toxicity:

As per the standard toxicity guidelines an acute toxicity study was carried out on female SD rats²⁵. The approach is based on fixed doses that are sufficiently spaced out to allow for the ranking of a chemical for classification and hazard assessment purposes. The rats were put on overnight fast before administration of test product. This was a two-step study and three rats were used in each step after one week of acclimatization to the circumstances of the animal house. In accordance with the recommendations, starting dose could be 5, 50, 300, or 2000 mg/kg as per the prior information available for a similar test product¹⁵. Since amla extract is a food supplement, we selected 2000 mg/kg dose for our study. Initially, 3 rats were administered with the test product via oral route using stainless steel canula. A veterinarian was assigned to monitor the toxicity symptoms and abnormal behavior of rats. The observation period was 2 weeks and rats were weighed on weekly basis.

In order to corroborate the results of the first phase, the following step involved 3 more animals and giving them 2000 mg/kg of amla extract, which did not result in any of the three animals dying or exhibiting abnormal signs and adverse effects. The animals were slaughtered after 14 days, and gross pathological abnormalities were noted.

Sub-chronic toxicity:

This toxicity investigation was conducted in accordance with standard guideline for testing of chemicals²⁶. As per the recommendations, minimum three doses of test product and a simultaneous control should be studied. The low, medium, and high doses were chosen to be 100 mg/kg, 500mg/kg and 1000mg/kg, respectively in rats. A total of one hundred SD rats (50 male and 50 female) were used in this study. The low, medium, high dose and control groups were containing 20 rats (10 male and 10 female) in each. Two recovery groups of 10 rats (5 male and 5 female) in each were assigned as ‘recovery control’ and ‘recovery high dose’ groups. Before the start of the dosing, the rats were acclimated for one week. Using a stainless-steel cannula, amla extract (suspended in 1% Tween 80) was given orally at dosages 100, 500, and 1000 mg/kg over the course of 90 days. Similar to this, the fourth group was given 1% Tween 80 as vehicle orally for the same period and was referred to as the control.

The animals in recovery groups received daily dose of vehicle (1 ml/kg) or the high dose (1000 mg/kg) of amla extract for 3 months. These rats were monitored by close supervision for an additional 14 days following the 90 days study period in order to check for any delayed symptoms of toxicity or reversibility of existing abnormal symptoms. Daily checks were made for the animals' appearance, behavior, and indicators of toxic exposure. A trained veterinary professional performed ophthalmologic examinations on all animals both at the start of the trial (baseline) and when it was over. At the conclusion of the study period (after 90 days), examination of grip strength and motor functions were conducted in accordance with published procedures^27-30. These assessments included sensory reactivity to stimuli of various types, including reflexes of various kinds and auditory, visual, and proprioceptive stimuli.

Throughout the study period, the animals were daily observed for any abnormal behavior, clinical symptoms of toxicity and mortality by a qualified veterinarian. At the end of the study, animals were fasted overnight and blood samples were drawn. A part of blood sample was used as such for hematological assessments whereas serum was separated from remaining blood sample for biochemical estimations. After blood collection, animals were euthanized and organs were collected carefully. Weight of all the organs was recorded after removal of any adherent tissue. The organs were stored in 10% formalin for histopathology assessments.

Statistical analysis:

GraphPad Prism software was used to do an analysis of variance (ANOVA) on the data. Following an ANOVA, Dunnett's pair-wise analysis was conducted for comparison of the mean values in amla extract and control groups. Mean±SD (standard deviation) was used to present the data and results between the groups were considered significant if p value was 0.05 or less.

RESULTS:

Acute toxicity in rats:

None of the rat showed any apparent symptom of toxicity at 2000 mg/kg dose of amla extract. Animals receiving the test product in steps I and II did not succumb to any deaths. Each rat's weight gain was consistent with normal levels (Table 1). At the end of the investigation, all animals were slaughtered and the organs were found normal in gross pathology. There was no abnormality on the body's external surface and all the orifices were normal according to the gross necropsy. The uterus, ovaries, thymus, pituitary glands, spleen and other key organs and tissues were all normal. According to the guidelines, the amla extract tested in this study may be considered as GHS (Globally Harmonized System) category 5. It indicates that LD50 is greater than 2000mg/kg in rats.

Sub-chronic toxicity study:

Clinical signs and death:

All the amla extract and control group animals completed the 90 days study period and there was no death reported in the study. Behavior of all the rats was normal as per the observations of veterinarian involved in the study. There was no clinical sign of toxicity observed in any of the control, treated or recovery group rats.

Table 2. Heamatological data of male rats after 90 days feeding of amla extract

Groups	Doses (mg/kg)	WBC (X 10³)	RBC (X 10⁶)	Hb (gm/dl)	PCV (%)	MCV (fL)	MCH (pg)	MCHC (g/dl)	Platelet count (X 10⁵)	RDW (%)	Clotting time (s)
Control	0	8.11± 0.91	7.12± 0.82	14.20± 1.64	46.28±4.72	53.14±6.28	17.26±1.53	31.54± 3.98	7.24± 0.88	17.22±1.82	126.20± 6.06
Low dose	100	8.61± 1.04	6.95± 0.91	14.19± 1.89	47.10±6.23	57.20±7.33	18.66±1.98	32.32± 3.31	6.34± 0.65	17.46±1.36	122.80± 3.56
Medium dose	500	8.23± 0.92	7.15± 1.03	15.02± 0.99	50.76±6.92	54.62±6.13	17.76±1.94	31.74± 3.82	6.19± 0.64	16.22±1.23	129.20± 7.19
High dose	1000	9.02± 0.99	7.34± 0.98	14.12± 1.04	49.16±6.39	54.34±8.31	19.16±1.92	31.70± 3.48	8.12± 0.78	19.02±0.98	121.60± 9.09
Control recovery	0	8.87± 1.02	8.01± 0.97	14.75± 1.58	47.72±7.93	54.64±7.43	17.78±1.76	33.92± 3.48	6.12± 0.78	18.96±1.68	125.40± 7.09
High dose recovery	1000	8.91± 0.78	6.94± 0.82	15.08± 1.86	46.66±5.64	55.60±6.14	18.22±1.81	32.14± 3.19	7.34± 0.99	18.16±1.89	121.40± 6.27

Values are Mean±SD. No significant differences (P>0.05) were observed between the test and control groups. Hb=Hemoglobin; PCV=Packed Cell Volume (Hematocrit); MCV= Mean Corpuscular Volume; MCH= Mean Corpuscular Hemoglobin; MCHC= Mean Corpuscular Hemoglobin Concentration; RDW= Red Blood Cell Distribution Width

Table 3. Heamatological data of female rats after 90 days feeding of amla extract

Groups	Doses (mg/kg)	WBC (X 10³)	RBC (X 10⁶)	Hb (gm/dl)	PCV (%)	MCV (fL)	MCH (pg)	MCHC (g/dl)	Platelet count (X 10⁵)	RDW (%)	Clotting time (s)
Control	0	7.48± 0.98	6.96± 0.93	14.50±1.47	44.48±5.44	57.40±2.75	17.98±1.35	30.60±4.34	7.67± 1.00	14.28±1.82	124.80± 8.20
Low dose	100	7.98± 1.64	7.37± 0.92	15.30±1.91	45.66±6.21	56.08±2.88	18.12±1.93	29.92±3.20	7.85± 0.77	16.20±1.64	128.80± 5.81
Medium dose	500	8.17± 1.47	8.54± 1.35	14.90±1.94	42.60±5.21	57.54±2.66	17.96±2.29	28.92±4.45	7.63± 1.08	15.46±1.38	130.00± 9.35
High dose	1000	7.37± 0.93	7.69± 0.96	14.77±1.82	47.76±6.54	58.16±2.22	18.02±1.97	27.94±3.70	6.87± 0.69	16.94±1.96	132.60± 4.62
Control recovery	0	7.85± 0.48	7.98± 0.99	13.63±1.79	48.82±6.93	56.18±1.25	17.66±2.02	30.10±3.65	7.94± 0.56	17.46±1.93	121.20± 4.60
High dose recovery	1000	8.12± 0.98	7.23± 1.43	14.17±1.34	45.42±6.48	57.70±2.58	16.92±1.91	31.10±3.13	7.47± 1.33	15.14±1.75	122.40± 7.37

Table 4. Biochemistry data of male rats after 90 days feeding of amla extract

Groups

Doses (mg/ kg)

TC (mg/dl)

TG (mg/dl)

ALP (IU/I)

Bil (mg/ dl)

Pro (g/dl)

SGOT (U/I)

SGPT (U/I)

BUN (mg/dl)

Creatinine (mg/ dl)

FBG (mg/ dl)

Control

64.60±

7.47

44.20±

4.71

129.20±

9.20

0.34±

0.05

7.56±

0.60

68.20±

8.23

47.40±

5.27

14.46±

1.93

0.32±

0.04

85.00±

8.37

Low dose

100

68.80±

3.77

38.60±

6.39

132.40±

9.24

0.42±

0.04

8.60±

0.82

70.40±

6.50

40.40±

4.45

16.40±

0.93

0.42±

0.04

89.20±

6.06

Medium dose

500

73.00±

7.25

42.40±

4.51

135.60±

6.54

0.42±

0.04

6.44±

1.07

81.40±

8.17

43.40±

7.50

13.90±

1.32

0.30±

0.07

96.40±

9.50

High dose

1000

70.00±

4.06

41.20±

5.36

124.40±

8.26

0.32±

0.04

6.78±

0.44

71.80±

9.58

42.40±

5.68

15.04±

1.89

0.34±

0.05

88.40±

8.26

Control recovery

74.60±

6.47

47.60±

6.31

131.60±

9.71

0.32±

0.04

6.96±

0.93

69.40±

8.29

41.80±

5.07

15.02±

1.37

0.30±

0.07

88.60±

7.54

High dose recovery

1000

70.80±

6.38

43.20±

4.76

138.80±

7.79

0.38±

0.04

7.16±

0.71

80.20±

7.29

38.80±

4.66

14.64±

1.35

0.32±

0.08

91.20±

4.76

Values are Mean±SD. No significant differences (P>0.05) were observed between the test and control groups. TC=Total cholesterol; TG=Triglycerides; ALP=Alkaline phosphatase; Bil=Total bilirubin; Pro=Total protein; SGOT=Serum glutamic oxaloacetic transaminase; SGPT=Serum glutamic pyruvic transaminase; BUN=Blood urea nitrogen; FBG=Fasting blood glucose

Table 5. Biochemistry data of female rats after 90 days feeding of amla extract

Groups	Doses (mg/ kg)	TC (mg/dl)	TG (mg/dl)	ALP (IU/I)	Bil (mg/ dl)	Pro (g/dl)	SGOT (U/I)	SGPT (U/I)	BUN (mg/dl)	Creatinine (mg/ dl)	FBG (mg/ dl)
Control	0	71.60± 8.08	48.80± 4.27	128.20± 7.85	0.40± 0.07	6.88± 0.90	72.40±7.44	41.40±4.16	14.90± 1.24	0.30± 0.07	92.20±9.04
Low dose	100	65.40± 5.41	44.20± 5.17	114.40± 8.35	0.42± 0.04	7.48± 0.85	66.40±7.83	44.40±7.16	16.52± 1.82	0.34± 0.05	88.20±5.36
Medium dose	500	81.40± 6.50	39.40± 3.21	125.40± 8.71	0.34± 0.05	7.90± 1.15	73.60±8.14	40.80±5.02	15.04± 0.94	0.42± 0.08	76.40±9.26
High dose	1000	77.40± 8.29	44.40± 6.27	132.60± 8.02	0.36± 0.09	7.02± 0.56	81.60±8.41	46.40±6.19	13.74± 1.15	0.32± 0.04	89.40±6.11
Control recovery	0	78.00± 5.05	42.60± 8.38	119.40± 9.09	0.32± 0.04	8.20± 0.89	78.40±8.88	44.60±5.32	14.30± 1.61	0.32± 0.04	86.20±6.46
High dose recovery	1000	72.40± 8.29	45.60± 5.22	132.60± 8.20	0.30± 0.07	7.48± 0.85	75.40±7.44	47.20±4.55	16.04± 0.92	0.32± 0.04	88.60±8.38

Relative organ weights and histopathology:

There was no significant difference in the relative weight of major organs between amla extract and control group of rats. The gross features of all the organs were also normal as per the examination by the veterinarian. The tissue sections observed under the microscope did not reveal any abnormal findings. Sections from kidney of all the rats showed normal cortex and medulla. There was no tubular necrosis or any sign of inflammation (Figure 1). The lobular architecture seen in the sections of liver was normal in amla extract treated rats. The hepatocytes were prominent and there was no necrosis of dilation of sinusoids (Figure 2). The glial cells and nerve cells were appeared normal in the section of brain in all the rats (Figure 3). Arrangement of spermatogonia and Sertoli cells was normal in the sections of testes. The seminiferous tubules were also in normal shape. The muscles of uterus were in perfect shape as observed by a qualified veterinary pathologist (Figure 4).

Figure 1. Kidney histopathology sections (X100). (A) Control male (B) Amla extract male (C) Control female (D) Amla extract female.

Figure 2: Liver histopathology sections (X100). (A) Control male (B) Amla extract male (C) Control female (D) Amla extract female.

Figure 3: Brain histopathology sections (X100). (A) Control male (B) Amla extract male (C) Control female (D) Amla extract female.

Figure 4: Testes (A&B) and uterus (C&D) histopathology (X100). (A) Control male (B) Amla extract male (C) Control female (D) Amla extract female.

DISCUSSION:

According to studies, the amla fruit extract has a number of beneficial properties, including antimutagenic³¹, hepatoprotective^32,33, anticarcinogenesis³⁴, antiulcerogenic³⁵, anti-tumor³⁶, antiproliferative activities³⁷ and induction of apoptosis³⁸. Although amla is being used for many different purposes, there is little information on its toxicity or safety when exposed repeatedly. According to OECD recommendations, the current study examined the single dose acute and 90-days repeated dose subchronic toxicity of amla extract in experimental rats.

The determination of acute oral toxicity in small animals is typically the first stage in the examination of the toxic properties of a test product. According to the study's findings on acute toxicity, rats can safely consume up to 2000mg/kg of amla extract without experiencing any adverse effects. Mokkhasmit et al. (1971) didn’t observe any toxic potential of an amla fruit extract prepared using ethanol as a solvent and fed to mice at 5000mg/kg dose, which is consistent with our findings³⁹. Since the amla extract utilized in this investigation had an LD50 > 2000mg/kg through acute exposure in rats, it is thought to be safe.

The repeated feeding of amla extract at maximum dose of 1000mg/kg to animals did not cause any death or abnormal behavior. The change in body weight over 90 days duration was normal and animals consumed food and water similar to control rats. As per the study published by Adeneye et al. (2006), the hematopoietic system is very susceptible to hazardous substances and is a significant indicator of the pathological and physiological well-being in humans as well as animals⁴⁰. Hematological measurements are crucial for determining the status of functions of bone marrow and conditions like hemolysis and anemia along with other intravascular effects. Because the hematological parameters were equal in the treated and control rats, it may be concluded that the amla extract is safe on rat hematopoiesis or leukopoiesis. In general, aberrant kidney, liver, and pancreatic function should be noticed whenever blood chemistry profile deviate in a significant manner as compared to the normal values⁴¹. In every rat given amla extract treatment, all biochemical indicators were within normal limits. This shows that the liver and kidneys of the treatment rats were in good condition. All of the major organs' relative organ weights were comparable among various test and control groups, and the results agreed with previously published research^42,43.

The findings of our safety study are somewhat similar and in line with a published safety study of dietary ALA-DAG oil repeated exposure in rats⁴⁴. In this study, special rat feed containing various percentage of ALA-DAG oil (1.38%, 2.7% and 5.50%) were administered to rats for three months. There were no signs of toxicity in any of the clinical observations, body weight, urine analysis, general behavior, blood profile, liver/kidney functions and histopathological findings. The data of control rats was comparable to the rats fed with the diet containing ALA-DAG oil. The authors found that maximum dose of ALA-DAG oil without toxic effects for male rats was 2916mg/kg whereas for female rats it was 3326mg/kg. In another study, ALA-DAG oil was non-toxic in a bacterial reverse mutation test (Ames test) and in vitro micronucleus test in the culture of Chinese hamster lung cells with and without metabolic activation, and in the in vivo bone marrow micronucleus test in mice⁴⁵. It was concluded that ALA-DAG oil is devoid of any genetic toxicity effects as tested conditions.

Traditionally, amla extracts are standardized with ascorbic acid, tannins (galloellagi tannins and hydrolysable tannins) or other polyphenols. The ALA-DAG present in the amla extract has tremendous benefits to human health specially to manage the heart health, visceral fat and metabolic disorders^21-23. In prior studies, amla has been examined for the possible management of dyslipidemia and safety of such formulations has also been studied^16,17. But the extract used in the present study is significantly different from the earlier studied products wherein only the fleshy part of the amla fruits was used. The whole fruit (including a hard seed) has been used in preparing the extract used in the present study and standardized with ALA and ALA-DAG.

The histopathology and study of gross lesions in the internal tissues was conducted to further confirm whether or not the internal organs/tissues had been damaged. However, no significant changes in the histology or gross observations were detected in the internal organs or tissues in any of the amla extract treated rats. The results of necropsy suggested that all of the examined organs treated with highest dose (1000 mg/kg) of amla extract were normal. Therefore, the No Observed Adverse Effect Level (NOAEL) of amla extract in rats was found as 1000mg/kg. The 1000 mg/kg in rats corresponds to about 11 g daily in human weighing 70 Kg as per the established dose conversion method⁴⁶. Assuming a 10-fold safety factor, this calculates to a NOAEL (human weighing 70 Kg) dose of 1100mg/day.

CONCLUSION:

The LD50 of amla extract (Tri-Low®) for rats is >2000 mg/kg as observed in the acute toxicity study. Repeated ingestion of amla extract for 90 days (sub chronic) to rats at the highest dose of 1000mg/kg did not result in any adverse effects. Therefore, it may be considered as NOAEL. Further evaluation of Tri-Low® for its cardiac benefits, management of obesity and other metabolic disorders can be planned owing to a large safety profile. To maintain the cardiac and metabolic health, it can be used as a daily food supplement without any concern of safety.

CONFLICT OF INTEREST:

The authors declare that there is no conflict of interest.

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Received on 28.11.2023 Modified on 18.04.2024

Research J. Pharm. and Tech 2024; 17(10):4887-4894.

DOI: 10.52711/0974-360X.2024.00752

Dose of amla extract (mg/kg)	Rat No.	Body weight (g)					No. of rats died
Dose of amla extract (mg/kg)	Rat No.	Initial	Day 7	Weight gain in one week	Day 14	Weight gain in two weeks	No. of rats died
2000	1	184	192	8	201	17	0
	2	182	192	10	202	20
	3	195	206	11	216	21
	4	180	189	9	200	20
	5	183	190	7	199	16
	6	190	199	9	209	19