**Norzahirah Ahmad \*, Bee Ping Teh, Siti Zaleha Halim, Nor Azlina Zolkifli, Nurulfariza Ramli and Hussin Muhammad**

Herbal Medicine Research Centre, Institute for Medical Research, National Institutes of Health, Ministry of Health Malaysia, Setia Alam, Shah Alam 40170, Malaysia; bpteh\_km@yahoo.com (B.P.T.); ctzaleha.h2@gmail.com (S.Z.H.); azlina.zolkifli@moh.gov.my (N.A.Z.); nurulfariza.r@moh.gov.my (N.R.); hussin.m@moh.gov.my (H.M.)

**\*** Correspondence: norzahirah.a@moh.gov.my

Received: 10 August 2020; Accepted: 31 August 2020; Published: 13 October 2020

**Abstract:** Coffee infused with the additive *Eurycoma longifolia*, also known as Tongkat ali (TA), has become widely available in the Malaysian market. Safety evaluations for consumption of the products have been called for due to the herbal addition. This study investigates the acute, subacute and chronic effects of a commercial TA coffee in Sprague Dawley rats when given in a single, repeated and prolonged dosage. The dosages of 0.005, 0.05, 0.30 and 2 g/kg body weight (BW) were used in the acute study and 0.14, 0.29 and 1 g/kg BW were used in the repeated dose studies. The in-life parameters measured were food and water intake, body weight and clinical observations. Blood were collected for hematology and clinical biochemistry analyses. All animals were subjected to full necropsies. Non-toxicity-related changes were observed in the food and water consumption parameters. Body weight showed normal increments and none of the animals had any clinical signs of toxicity. Microscopically assessed organ tissues did not reveal any abnormalities. There was significant decrease of platelet count in all the chronic study male treated groups. Significant elevation of renal profile parameters in both gender groups given 0.29 g/kg BW, along with liver and lipid profile elevation in some female groups of the chronic study were noted. No dose-dependent relationship was apparent in the dosage range tested, though these changes may suggest an initial safety indication to the TA coffee. The study concludes that the no observed adverse effect level (NOAEL) for this commercial TA coffee was 1 g/kg BW.

**Keywords:** *Eurycoma longifolia*; Tongkat ali; toxicity; infused coffee; herbal additives

### **1. Introduction**

*Eurycoma longifolia,* also known as Tongkat ali (TA), is a native plant to Southeast Asian rain forests. The roots are believed to boost wellness while having aphrodisiac, anti-malarial and other therapeutic properties, such as anti-inflammatory and anti-osteoporosis [1–8]. TA has received considerable attention among Malaysian consumers and is traditionally processed into a drink, although coffee makers also tout it as a healthy additive in coffee drinks. Safety information such as the no observed adverse effect level (NOAEL) for *E. longifolia* in its aqueous extract form has been previously reported to be more than 3 g/kg in mice [9] and more than 1 g/kg and 5 g/kg in rats in two separate studies [10,11], while the powdered form of the root was reported to have an acute limit dose of more than 6 g/kg [12]. Although these studies reported a high tolerance of the highest dose used, they also reported hydropic liver histology indicating hepatotoxicity [11] and minor yet significant hematology and clinical biochemistry changes [10,12]. Additionally, information for TA extract long-term consumption is limited and its use as an herbal additive in food products has never been evaluated and warrants further investigation.

Worldwide, the legislation with regards to beverages with herbal additives varies between countries [13], whereas currently in Malaysia, safety testing for food products in this category has not been accounted under the Food Act (1983), Ministry of Health, Malaysia and Food Regulation (1985), Ministry of Health, Malaysia [14,15]. While there have been no claims of ill health and the existing legislation in Malaysia does not require safety testing for using *E. longifolia* extracts in coffee, the World Health Organization (2004) recommends herbal medicinal ingredients in products to be assessed for its safety [16]. Furthermore, the addition of therapeutically claimed traditional ingredients have clouded the definitions between the food and drug regulations. It is often unclear which testing standards are required and the sets of regulations to abide by for the herbal additives in food products [17–19]. Consequently, without safety testing, marketing of such coffee outside of Malaysia is restricted due to strict import regulations with various levels of evidence requirements.

Safety testing adds value to the product, elevates the product safety information, improve consumer trust and may prevent potential incidents such as interaction between pharmaceutical drugs and herbal-incorporated food products, where occurrence may have gone unnoticed [17,18,20,21]. The aim of this paper is to investigate the safety of a commercial TA coffee, with reference to the Organization for Economic Cooperation and Development (OECD) testing guidelines 420, 407 and 452 [22–24]. The TA coffee was administered to Sprague Dawley rats in a single-dose 14-day acute study and as a repeated daily dose for 28 days and 6 months in the subacute and chronic toxicity studies, respectively.

#### **2. Materials and Methods**

#### *2.1. Test and Reference Item Preparation*

The commercial GTHerb Gold Coffee *E. longifolia*-mixed coffee was provided by GTHerb Industries Sdn. Bhd. Analysis of the content was done by Als Technician and Technology Park Malaysia, Biotech Sdn. Bhd. (Kuala Lumpur, Malaysia). The TA coffee (test item) was physically in powdered form and dark brown in color. Prior to the daily preparation, the test item was kept at temperatures between 25 and 28 ◦C. The test item prepared was calculated based on the individual rat s body weight and according to the required dosage. The test item for rats weighing less than 100 g was dissolved in 1 mL hot distilled water, whereas for rats weighing 200 g and above, 2 mL of hot distilled water was used. The reference item (distilled water) was administered in the same manner. The administered amount of test item was adjusted weekly to correspond with the weekly change in the body weights.
