Do Testosterone Booster Supplements Really Work?


A progressive decline in testosterone (T) is seen with male aging, estimated at 0.4% to 2.0% decline per year after age 30 [1]. It is estimated that men in their 70s have mean T levels 35% lower than younger men [2].

The progressive decrease in serum T with aging is a well-documented progress. TRT is a well-established treatment option for those with symptomatic T deficiency. TRT has a well-defined risk profile, with clear guidelines for indications, contraindications, dosing, treatment goals, and monitoring during treatment [11].

However, despite the FDA approved status of exogenous T, a recent review looking at the quality of online information related to TRT found that online information is incomplete, often failing to describe important safety information and the need for regular monitoring [12].

Herbal supplements designed to increase T are poorly studied yet remain popular among aging men who seek to increase their T without standard TRT. There is often the hope that supplements will increase T in a more “natural” manner, and therefore be free from risk. However, this does not appear to be the case. These products are often poorly regulated and minimally studied, both in terms of efficacy and toxicity profile.

For the T booster supplements surveyed, 109 components were identified. There were a mean 8.3 components per supplement (range, 29; median, 7; mode, 6; standard deviation, 5.52; interquartile range, 1–7).

The most common components for the supplements were zinc, fenugreek, vitamin B6, Tribulus, magnesium, boron, diindolemethane, Eurycoma longifolia, and Maca extract.

A full list of the individual components, with percent of supplements containing these components, is found in Table 1.

Table 1

Individual components for “T booster” supplements and the percentage of surveyed supplements containing those components

SupplementSupplements containing the component (n=45)
Zinc29 (64.4)
Fenugreek extract22 (48.9)
Vitamin B620 (44.4)
Tribulus19 (42.2)
Magnesium17 (37.8)
Boron12 (26.7)
Diindolemethane12 (26.7)
Eurycoma longifolia extract12 (26.7)
Maca extract10 (22.2)
D-Aspartic acid9 (20.0)
Ashwagandha extract8 (17.8)
Vitamin D8 (17.8)
Black pepper fruit extract7 (15.6)
Mucuna purines7 (15.6)
Tongkat ali extract7 (15.6)
Vitamin B127 (15.6)
L-Arginine6 (13.3)
Vitamin B36 (13.3)
Vitamin B96 (13.3)
Calcium5 (11.1)
Ginseng5 (11.1)
Nettle root extract5 (11.1)
Saw palmetto5 (11.1)
Selenium5 (11.1)
Shilajit5 (11.1)
Avena sativa extract4 (8.9)
Bioperine4 (8.9)
Damiana leaf4 (8.9)
Epimedium4 (8.9)
Ginkgo biloba4 (8.9)
Rhodiola rosea extract4 (8.9)
Grape extract3 (6.7)
Horney goat weed extract3 (6.7)
Safed musli extract3 (6.7)
Agaricus bisporus fruit extract2 (4.4)
Akarkara extract2 (4.4)
Alpha-Glycerolphosphorylcholine2 (4.4)
Astragalus root powder2 (4.4)
Broccoli2 (4.4)
Bulbine natalensis2 (4.4)
Caffeine2 (4.4)
Cnidium monnieri2 (4.4)
Coleus extract2 (4.4)
Dehydroepiandrosterone2 (4.4)
Green tea leaf extract2 (4.4)
L-Carnitine2 (4.4)
L-Citrulline2 (4.4)
Laxosterone2 (4.4)
Melatonin2 (4.4)
Resveratrol2 (4.4)
Rosemary extract2 (4.4)
Vitamin E2 (4.4)
Yohimbe extract2 (4.4)
Agmatine1 (2.2)
Aminoethanesulfonic acid1 (2.2)
Anacyclus pyrethrum1 (2.2)
Androsta1 (2.2)
Apigenin1 (2.2)
Apple extract1 (2.2)
Calcium fructoborate1 (2.2)
Calcium glucarate1 (2.2)
Chrysin1 (2.2)
Eleutherococcus senticosus1 (2.2)
Epicatechin1 (2.2)
Fadogia agrestis1 (2.2)
Fumarate1 (2.2)
GABA1 (2.2)
Garlic extract1 (2.2)
Glycyrrhiza glabra (licorice)1 (2.2)
Hesperidin1 (2.2)
HTP (5-HTP)1 (2.2)
Huperzia serrata extract1 (2.2)
Indole-3-carbinol1 (2.2)
Kava1 (2.2)
L-Histidine1 (2.2)
L-Phenylalanine1 (2.2)
L-Theanine1 (2.2)
L-Tyrosine1 (2.2)
Longjack extract1 (2.2)
Medium chain triglyceride oil1 (2.2)
Methylliberine1 (2.2)
N-MDA1 (2.2)
Orchic substance1 (2.2)
Oyster extract1 (2.2)
Panax notoginseng1 (2.2)
Pantothenic acid1 (2.2)
Phosphorus1 (2.2)
Phytosterol1 (2.2)
Piper nigrum1 (2.2)
Pyrroloquinoline quinone1 (2.2)
Prolinsis1 (2.2)
Prunella vulgaris1 (2.2)
Quercetin1 (2.2)
Red clover1 (2.2)
S-2-Amino-5-guanidinopentanoic acid1 (2.2)
Sarsaparilla extract1 (2.2)
Schizonepeta1 (2.2)
Sodium alpha lipoic acid1 (2.2)
Succinate1 (2.2)
Theobroma cacao1 (2.2)
Trigonella foenum1 (2.2)
Turmeric1 (2.2)
Valerian root1 (2.2)
Vitamin B21 (2.2)
Vitamin C1 (2.2)
Vitamin K1 (2.2)
White tea extract1 (2.2)
Wild yam extract1 (2.2)
Withania somnifera1 (2.2)
Values are presented as number (%).
GABA: gamma-aminobutyric acid, HTP: hydroxytryptophan, N-MDA: N-Methyl-D-aspartate.

Table 2

“Beneficial” claims made by supplements

“Beneficial” claimSupplements claiming these benefits (n=50)
Boost T or free T45 (90.0)
Build body lean mass or muscle mass31 (62.0)
Increase sex drive or libido25 (50.0)
Feel or be stronger24 (48.0)
Increase energy15 (30.0)
Burn fat or prevent fat build up14 (28.0)
Reduce recovery time14 (28.0)
Anti-estrogen effect13 (26.0)
Improve sleep10 (20.0)
Better mood5 (10.0)
Increase growth or luteinizing hormone5 (10.0)
Reduced cortisol4 (8.0)
Improve erections3 (6.0)
Improved vascularity2 (4.0)
Promote healthy aging1 (2.0)
Improve bones and joints1 (2.0)
Values are presented as number (%).
T: testosterone.

Illustrating this, a 2017 case report described new-onset, bilateral pulmonary embolisms secondary to over-the-counter fenugreek-extract-containing T supplements [13]. In 2014, the FDA issued a general warning for the risk of venous blood clots associated with T product use [14]. While not specified in the FDA warning, this potential risk may extend to herbal T supplements as well.

Another study looking at the effect of red clover on the quality of life, and sexual function in men found that this supplement did not change sexual or erectile function, and resulted in a significant increase in liver transaminases [15]. In short, these vitamin and herbal substances may not be as safe as the public perceives them to be.

In this study we sought to evaluate the composition of “T booster” supplements, their advertised claims, and we compared this with the published literature and RDA. In general, the available literature supporting the claims made by the supplements was often sparse or non-existent. Only 5.5% of supplements had more than two studies looking at their effect on T. Supplements 61.5% had no data looking at their effect on T.

For 24.8%, or 27 out of 109 individual supplements, there was data showing an increase in T with supplementation. However, for many of these (15 of the 27, or 55.5%) there was also conflicting data showing no change or a decrease in T with supplementation. Of concern, 10.1% of components had published data that found a decrease in T with supplementation.

It is unclear why companies would include components in their supplements that have no evidence to support their use. However, it is even more concerning that some of these supplements may in fact decrease serum T.

Regarding the results of our PubMed review, there were no studies looking at the effect of the individual supplements on T levels for 67 of the supplements (61.5%). For 19 supplements (17.4%) there was a single study looking at the effect of supplementation on T. For 13 supplements (11.9%) there were 2 studies; for 3 supplements (2.8%) there were 3 studies; for 4 supplements (3.7%) there were 4 studies; for 1 supplements (0.9%) there were 5 studies; for 1 supplements (0.9%) there were 6 studies.

For 27 individual supplements (24.8%), there was data showing an increase in T with supplementation. Eleven individual supplements (10.1%) had data showing a decrease in T with supplementation, and 20 individual supplements (18.3%) had data showing no change in T with supplementation. Given these findings, 15 individual supplements (13.8%) therefore had conflicting data regarding their effect on T. These data are summarized in Table 3.

Table 3

Published evidence showing an increase, decrease or no change in testosterone (T) with supplementation

SupplementIncrease TDecrease TNo change in TConflicting data
Anacyclus pyrthrumX
Ashwagandha extractXXX
Bulbine natalensisX
D-Aspartic acidXXXX
Eurycoma longifolia extractX
Fenugreek extractX
Ginkgo bilobaX
Glycyrrhiza glabraXXX
Green tea leaf extractXXX
Horney goat weed extractX
Maca extractXXX
Mucuna purineX
Pantothenic acidX
Red cloverX
Rhodiola rosea extractX
Rosemary extractX
Saw palmettoX
Tongkat ali extractX
Vitamin B6X
Vitamin DXXX

RDA information was not available for many of the individual components for the “T boosters”. The FDA does not issue RDA and upper tolerable limit data for herbal supplements. Supplements contained a median 1,291% of the RDA for vitamin B12, 807.6% of vitamin B6, 272% of zinc, 200% of vitamin B5, and 187.5% of vitamin B3 (Table 4).

Table 4

Recommended Daily Allowance (RDA) and upper tolerable limit for each of the individual supplement components (when available)

Supplement (vitamins & minerals)Containing (%)RDAUpper limitRangeMedian doseCompared to median (%)
Calcium11.11,000 mg2,500 mg11–175 mg31.5 mg3.1
Folate13.3400 μg1,000 μg100–800 μg400 μg100.0
Magnesium37.7320 mg350 mg9–450 mg450 mg140.6
Phosphorus2.2700 mg4,000 mg135 mg135 mg19.2
Selenium11.155 μg400 μg50–70 μg50 μg90.9
Vitamin B22.21.3 mgNA2 mg2 mg153.8
Vitamin B311.116 mg35 mg15–41 mg30 mg187.5
Vitamin B52.25 mgNA10 mg10 mg200.0
Vitamin B644.41.3 mg100 mg2–50 mg10.5 mg807.6
Vitamin B1215.52.4 μgNA6–500 μg31 μg1,291.0
Vitamin C2.290 mg2,000 mg90 mg90 mg100.0
Vitamin D17.7600 IU4,000 IU400–3,000 IU1,000 IU166.6
Vitamin E4.433 IU (synthetic)1,100 IU (synthetic)30–50 IU40 IU121.2
Vitamin K2.2120 μgNA50 μg50 μg41.6
Zinc64.411 mg40 mg1.05–50 mg30 mg272.0
NA: “not available” as there is no RDA or upper tolerable limit for these compounds.

It is worrisome that two supplements had greater than the UL of zinc. Relatively low levels of zinc over-supplementation have been shown to interfere with the utilization of copper and iron and to adversely affect high-density lipoprotein cholesterol concentrations. Slightly higher doses have been shown to result in anemia and neutropenia, as well as impaired immune function [16].

According to the 2018 American Urological Association Guidelines on the Evaluation and Management of T Deficiency, patients should be informed that T therapy may improve in erectile function, low sex drive, anemia, bone mineral density, lean body mass and/or depressive symptoms.

While TRT may have these effects, the FDA states that “Unlike drugs, supplements are not intended to treat, diagnose, prevent, or cure diseases. Claims like these can only legitimately be made for drugs, not dietary supplements.” [8]. However, despite this FDA statement, the “T booster” supplements made a host of claims. While some of these may be associated with improvements in T, others may not.

In addition, supplements also made promises for which there is limited to no available data for, including: to “feel or be stronger”, “increase energy”, “burn fat or prevent fat build up”, “reduce recovery time”, “improve sleep”, “improved vascularity”, “promote healthy aging”, and “improve bones and joints”. It is important that patients have a realistic picture of what to expect with T supplementation. Certainly, it is not a substitute for a healthy lifestyle, which is what many of the claims seem to tout.

Limitations of our study are that we used only Google as our search engine, and there may be regional and geographic differences in search engine results. We chose Google as our search engine because it is the most commonly used search engine [9], however, the addition of other search engines (Yahoo or Bing) may yield different results.

In addition, only the first and most frequently appearing 50 supplements were included in the study, acknowledging that other products and supplements may be available that were not studied here. This study also only utilized a single search term (“Testosterone Booster”).

While different search times produced results that were not relevant to this study, such as products with exogenous T and hormones, a different search phrase may have produced a varied supplement list. However, despite these limitations, our data clearly demonstrates the unrealistic expectations that are stated online for the role of vitamins and antioxidants in male infertility. This highlights the need for evidence-based patient education materials relating to this topic.

5 Eurycoma longifolia Jack (Tongkat Ali):

Eurycoma longifolia Jack (known as tongkat ali), a popular traditional herbal medicine, is a flowering plant of the family Simaroubaceae, native to Indonesia, Malaysia, Vietnam and also Cambodia, Myanmar, Laos and Thailand. E. longifolia, is one of the well-known folk medicines for aphrodisiac effects as well as intermittent fever (malaria) in Asia.

Decoctions of E. longifolia leaves are used for washing itches, while its fruits are used in curing dysentery. Its bark is mostly used as a vermifuge, while the taproots are used to treat high blood pressure, and the root bark is used for the treatment of diarrhea and fever. Mostly, the roots extract of E. longifolia are used as folk medicine for sexual dysfunction, aging, malaria, cancer, diabetes, anxiety, aches, constipation, exercise recovery, fever, increased energy, increased strength, leukemia, osteoporosis, stress, syphilis and glandular swelling.

The roots are also used as an aphrodisiac, antibiotic, appetite stimulant and health supplement. The plant is reported to be rich in various classes of bioactive compounds such as quassinoids, canthin-6-one alkaloids, β-carboline alkaloids, triterpene tirucallane type, squalene derivatives and biphenyl neolignan, eurycolactone, laurycolactone, and eurycomalactone, and bioactive steroids.

Among these phytoconstituents, quassinoids account for a major portion of the E. longifolia root phytochemicals. An acute toxicity study has found that the oral Lethal Dose 50 (LD50) of the alcoholic extract of E. longifolia in mice is between 1500–2000 mg/kg, while the oral LD50 of the aqueous extract form is more than 3000 mg/kg.

Liver and renal function tests showed no adverse changes at normal daily dose and chronic use of E. longifolia. Based on established literature on health benefits of E. longifolia, it is important to focus attention on its more active constituents and the constituents’ identification, determination, further development and most importantly, the standardization. Besides the available data, more evidence is required regarding its therapeutic efficacy and safety, so it can be considered a rich herbal source of new drug candidates. It is very important to conserve this valuable medicinal plant for the health benefit of future generations.

Evidenced-Based Pharmacology

5.1. Male Fertility Enhancement Effect

Infertility is a major clinical problem, which affects the people medically, economically and psychosocially. Almost, 15% of all couples in the U.S. are infertile, and it is predicted that the male factor is responsible in many of such cases [104]. Male infertility refers to a male’s inability to achieve a pregnancy in a fertile female. In humans, this accounts for 40%–50% of infertility cases [105,106]. Infertility in males is a multifactorial disease, based on numerous factors including reduced spermatogenesis and also production of dysfunctional sperm, which are the major prevalent underlying characteristic in idiopathic male infertility cases [107,108]. One meta-analysis of sixty-one studies worldwide reported s downward trend in the sperm count and semen volume over the past fifty years [109,110].

Mostly, the water-soluble E. longifolia extracts were reported to be able to enhance male fertility (with regards to higher semen volumes, spermatozoa count, and motility) in rodents [111,112] and in human trials [86,113,114].

The standardized extract F2 of E. longifolia (25mg/kg p.o) and its major quassinoids, especially eurycomanone (250 mg/kg p.o) improved rat spermatogenesis by affecting the hypothalamic-pituitary-gonadal axis and the potential efficacy may be worthy of further investigation [111].

Eurycomanone, the major quassinoid in the E. longifolia root extract, significantly increased testosterone production on a dose-dependent manner at 0.1, 1.0 and 10.0 μM (p < 0.05). It enhanced testosterone steroidogenesis at the rat testicular Leydig’s cells by inhibiting aromatase conversion of testosterone to oestrogen, and may also involve in phosphodiesterase inhibition at a high concentration, so authors have suggested that quassinoids from E. longifolia may be worthy for further development as new phytomedicines for the treatment of testosterone-deficient idiopathic male infertility and sterility [112]. Also, standardized extracts of E. longifolia Jack containing a high concentration of quassinoids (20% eurycomanone and 4% of 13α,21-dihydroeurycomanone) may have potential anti-estrogenic effects [86].

The quassinoid-containing E. longifolia extract affects male infertility by suppressing α-2-HS glycoprotein expression, which indirectly increases the testosterone levels and insulin sensitivity. They indicated that serum α-2-HS glycoprotein was reduced in rats treated with standardized E. longifolia extract, which will provide rational for further investigation in animal models of infertility with diabetes [113].

A randomized, double-blind, placebo-controlled, parallel group study was conducted to investigate the aphrodisiac clinical evidence of E. longifolia extract in men. The total twelve weeks’ study in 109-men between 30- and 55- years of age, divided in a group of 300 mg of water extract of E. longifolia (Physta®)-treated and placebo. The E. longifolia group showed higher scores in the overall erectile-function-domain (IIEF, p < 0.001), the sexual libido (14% by week 12), Seminal Fluid Analysis (SFA)-with sperm motility at 44.4%, and semen-volume at 18.2% after treatment [114].

Chan et al., statistically analyzed the spermatozoa count, morphology, motility, plasma testosterone level and Leydig cell count of the animals by ANOVA. Their results showed that the sperm counts of rats given the standardized methanol extract alone at doses of 50, 100 and 200 mg/kg were increased by 78.9%, 94.3% and 99.2%, respectively, when compared with that of control (p < 0.01) [115].

Ang and Ngai showed that the fractions of E. longifolia Jack (0.5 g/kg) decreased the hesitation time. Furthermore, they caused a transient increase in the percentage of the male rats responding to the right choice; more than 50% of the male rats scored “right choice”; using the electrical copulation cage [116].

E. longifolia has been shown to elevate serum testosterone and increased muscle strength in humans. Chen et al., investigated the effects of standardized water extract of E. longifolia (Physta®) at a dose of 400 mg/day for 6 weeks on testosterone: epitestosterone (T:E) ratio, liver and renal functions in male recreational athletes found no significant difference between the results of supplementation results and placebo [117].

Study on the sexual qualities of middle-aged male rats after dosing with 0.5 g/kg of various fractions of E. longifolia, showed that it enhanced the sexual qualities of the middle-aged male rats by decreasing their hesitation time as compared to controls [118].

A randomized, double-blind, study with placebo-controlled was conducted for proprietary freeze-dried water extract of E. longifolia (Physta®) effects on sexual performance and well-being in men. For this study, men aged 40–65 years were screened for 12-week. Results showed the significant improvements in scores for the sexual intercourse attempt diary, erection hardness scale, sexual health inventory of men, and aging male symptom scale (p < 0.05 for all), concluded that Physta® was well tolerated and more effective than placebo in enhancing sexual performance in healthy volunteers [59].

E. longifolia extract acts as a potential agent to increase spermatogenesis and sperm counts, and for reversing the effects of estrogen in rats, after fourteen consecutive days of treatment [119].

In other study, Ang et al., showed that E. longifolia produced a dose-dependent, recurrent and significant increase in the episodes of penile reflexes as evidenced by increases in quick flips, long flips and erections of the treated male rats during 30 min observation period [17].

According to Tambi and Imran’s investigations, 350 patients were given 200 mg of the E. longifolia extract daily, and follow-up semen analyses were performed every 3 months up to 9 months. These patients showed significant improvement in all semen parameters, allowing for 11 (14.7%) spontaneous pregnancies [120].

Erasmus et al., treated semen samples with E. longifolia extract (in vitro condition), found a significant dose-dependent trends for vitality, total motility, acrosome reaction and reactive oxygen species-positive spermatozoa; but no deleterious effects on sperm functions at therapeutically used concentrations (<2.5 µg mL−1) [121].

An increase in sperm count, motility and viability in rats, when treated with aqueous E. longifolia extract. Noor et al., investigated that E. longifolia can increase sexual behavior of male rats and the sperm quality; which were found to be dose dependent [122]. One study indicates that E. longifolia exerts proandrogenic effects that enhance the testosterone level [123].

The in vivo effect of aqueous extract of E. longifolia was investigated on body and organ weight as well as functional sperm parameters in terms of safety and efficacy in the management of male infertility, in male rats. Testosterone concentration increased by 30.2%, total sperm concentration, progressive motility and vitality significantly increased, MMP improved markedly by 25.1%, with increased in muscle weight, non-significantly, so it appears that E. longifolia use is safe for possible treatment of male infertility and ageing male problems [124].

In human studies, Tambi et al., treated a group of patients suffering from late-onset hypogonadism (LOH) with Tongkat ali extract, which showed significantly (p < 0.0001) improved the Ageing Males’ Symptoms (AMS) score as well as the serum testosterone concentration. Thus, Tongkat ali extract appears to be useful as a supplement in overcoming the symptoms of LOH and for the management of hypogonadism [125].

The testosterone deficiency syndrome (TDS), can be characterised by numerous symptoms, including low libido, fatigue, increased fat mass, osteoporosis or erectile dysfunction, and up-to 80% of men have experience some sort of ageing male symptoms. Conventionally, TDS is treated with testosterone replacement therapy (TRT). With the beneficial effects of this therapy, significant adverse effects have been indicated, including prostate cancer. E. longifolia is the herbal alternative to TRT, which has been shown to successfully restore serum testosterone levels, and significantly improve the physical condition and sexual health of patients. Therefore, E. longifolia might be considered a safe alternative to TRT [126].

For the copulatory activity of sexually sluggish rats, with acute (500, and 1000 mg/kg) and also subacute treatments with E. longifolia root powder, significantly reduced ejaculation latencies, and increased the percentage of mounting and ejaculating animals; while the subacute administration reduced post-ejaculatory interval. In case of impotent rats, both treatments increased the percentage of mounting and ejaculating rats. Serum testosterone levels were increased in rats that were treated subacutely, in comparison with control [127].

One experiment by Ang and Sim showed that E. Iongifolia Jack continued to enhance and also maintain a high level of both the total number of successful crossovers, mountings, intromissions and ejaculations during the 9–12th week observation period [128].

In animal research, an herbal combination containing Panax quinquefolius, Eurycoma longifolia, Epimedium grandiflorum, Centella asiatica, and flower pollen extracts enhanced erectile function [129]. Improvements were noted in the penile erection index (PEI). In boars, an herbal preparation containing Eurycoma longifolia, Tribulus terrestris, and Leuzea carthamoides increased libido (by 20%) and semen quality (volume, concentration, etc.) [130].

Randomized controlled trials investigating E. longifolia compared to placebo were included by Kotirum et al. and suggests that E. longifolia root extract may have a clinical benefit on improving erectile dysfunction performance. Based on current evidence, the herbal extract of E. longifolia may have clinical effect on erectile function, but needs further clinical evidence of efficacy trials to make any firm recommendation [131].

In a pilot study, Henkel et al. investigated the ergogenic effect of E. longifolia in elderly people and found that it is a potential herbal supplement for physically active aged male and female (age 57–72 years). Treatment resulted in significant increases in total and free testosterone concentrations and muscular force in men and women, when E. longifolia extract 400 mg/day was used for 5 weeks [132].

5.2. Antimalarial Effect

The WHO estimates that in 2013, there were 207 million annual cases of malaria, resulting in 627,000 deaths, from Plasmodium falciparum [133,134]. There are about 10,000 malaria cases per year in Western Europe, and 1300–1500 in the United States [135]. E. longifolia extract is traditionally used for malarial fevers and has good anti-malarial effect against P. falciparum.

Chan et al., tested the extracts of E. longifolia for antiplasmodial activity against a multi-drug resistant Thailand’s strain (K-1) of P. falciparum under in vitro conditions. They isolated 10-hydroxycanthin-6-one, eurycomalactone, eurycomanone and eurycomanol from the plant, which showed antimalarial activities [60].

According to Kardono et al., two compounds, eurycomanone and 7-methoxy-β-carboline-1-propionic acid showed significant antimalarial activity against P. falciparum strains [61]. Low et al., concluded that the administration of the bioactive standardized extract Fr2 (200 mg/kg) showed a good antimalarial effect. 13α(21)-epoxyeurycomanone and eurycomanone may be the only quassinoids contributing to the overall antimalarial activity of E. longifolia [62].

In study, conducted during 2008 in Mae Sot, Tailand, a standardized extract of E. longifolia containing three major quassinoids, eurycomanone (1), 13,21-dihydroeurycomanone (2) and 13α(21)-epoxyeurycomanone (3) was evaluated for antiplasmodial activity against Plasmodium falciparum. Activity was compared with that of artemisinin, using thirty-eight fresh parasite isolates and assessment of inhibition of schizont maturation. The IC50, IC90 and IC99 values for artemisinin were 4.30, 45.48 and 310.97 μg/L, and those for the root extract from E. longifolia 14.72, 139.65 and 874.15 μg/L respectively. The inhibitory activity of the E. longifolia extract was higher than that expected from the three quassinoids isolated from the plant, suggesting synergism between the quassinoids or the presence of other unidentified compounds [63].

Ang et al., tested E longifolia extract activity in vitro on Malaysian chloroquine-resistant Plasmodium falciparum culture. They showed that the antimalarial activity of E. longifolia Jack was dose-dependent and reached a maximum of <50% at 0.07−5.00 μg·mL−1 after 1-day post-treatment. However, complete inhibitions were observed at 1.25–5.00 μg·mL−1 extract after 3 days’ post-treatment and 0.62 and 0.31 μg·mL−1 after 4 and 6 days’ post-treatment, respectively [64].

E. longifolia methanol extract (TA164) decreased the glutathione (GSH) content of both infected and healthy erythrocytes at a certain dosage and incubation period. Both effects of TA164 to GSH content of host or parasite can be the cause of P. falciparum growth inhibition in vitro and screening the activity of GSH synthesis can be one of the procedures in evaluating the antimalarial properties of herbal products [136].

5.3. Cytotoxic and Anti-Proliferative Effect

Cytotoxic effects of novel drug entities and traditional medicines are very essential to be investigated before testing their further pharmacological activity. After establishment of positive cytotoxic effects, anti-proliferative effects (rate of cytotoxicity) are also investigated to check and confirm their further anti-cancer effectiveness, using in vitro as well as in vivo models. Various constituents from E. longifolia have been tested for cytotoxic effects, and some of these also showed positive anti-proliferative effects.

Cancer, medically known as a malignant neoplasm, is a broad group of diseases involving unregulated cells. In malignant neoplasm (cancer), cells divide and grow uncontrollably, forming malignant tumors, and invading nearby parts of the body. It may also spread to more distant parts of the body through the lymphatic system or bloodstream.

Over 200 different known cancers that can affect humans; and there are over sixty different organs in the body where a cancer can develop. A statistical report in 2012 showed that total 338,623 people were diagnosed with cancer in the UK, while 161,823 deaths from cancer ocurred (survival rate was 50%) [137].

E. longifolia has cytotoxicity and antiproliferative effects on various human cancer cell lines, as well as various solid tumors, including lung, breast and cervical cancers. Kuo et al., [36] isolated and identified nearly 65 compounds from the roots of E. longifolia and screened them for the potential cytotoxicity and anti-HIV activities by in vitro assays.

Among the compounds evaluated, 13β,21-dihydroxyeurycomanol [60], 6-dehydroxylongilactone [72], 9-methoxycanthin-6-one [75], canthin-6-one [76], eurylene [53], 9-hydroxycanthin-6-one [76], longilactone [75], 9-methoxycanthin-6-one 3N-oxide [76], 14,15β-dihydroxyklaineanone [75], pasakbumin C [50], canthin-6-one 9-O-β-glucopyranoside [76], were screened for in vitro cytotoxicity against A-549 and MCF-7 tumor cell lines [138] and no inhibition of HIV replication in H9 lymphocytes except for eurylene and pasakbumin B [139].

Compounds longilactone, 6-dehydroxylongilactone, 9-methoxycanthin-6-one, canthin-6-one, longilactone, 9-methoxycanthin-6-one, 14,15β-dihydroxyklaineanone, pasakbumin C, and canthin-6-one 9-O-β-glucopyranoside demonstrated strong cytotoxicity toward A-549 cell lines, however, longilactone, 6-dehydroxylongilactone, 9-methoxycanthin-6-one, eurycomanone, pasakbumin B, and 9-methoxycanthin-6-one displayed strong cytoxicity toward the MCF-7 cell line.

According to Park et al., [51] the compounds eurycomalactone [49], longilactone [140], and 14,15β-dihydroxyklaineanone [140] showed significant cytotoxicity in both A549 and MCF-7, while 13,21-dihydroeurycomanone [140] was more selective against A549 and eurycomanone [140] showed cytotoxic effects only against MCF-7. In the HeLa cell line, compounds eurycomalactone, 13,21-dihydroeurycomanone, eurycomanone, 13α(21)-epoxyeurycomanone, longilactone, and 14,15β-dihydroxyklaineanone displayed significant cytotoxicity showing the relative cell viability ranging from 21.01% ± 2.46% to 66.9% ± 6.67% at the concentration of 100 μM.

Three new [n-pentyl β-carboline-1-propionate, 5-hydroxymethyl-9-methoxycanthin-6-one, and 1-hydroxy-9-methoxycanthin-6-one] and 19 known β-carboline alkaloids were isolated from the roots of E. longifolia. These compounds were screened for in vitro cytotoxic activities; in which 9-methoxycanthin-6-one and canthin-6-one demonstrated significant cytotoxicity against human lung cancer (A-549) and human breast cancer (MCF-7) cell lines [76].

Kardono et al., isolated and characterized five cytotoxic constituents from the roots of E. longifolia. Four of the canthin-6-one alkaloids, namely, 9-methoxycanthin-6-one, 9-methoxycanthin-6-one-N-oxide, 9-hydroxycanthin-6-one, and 9-hydroxycanthin-6-one-N-oxide and one quassinoid, eurycomanone, were found to possess cytotoxic effects against a panel of cell lines like: human cancer cell types (breast, colon, fibrosarcoma, lung, melanoma, KB, and KB-V1) and murine lymphocytic leukemia (P-388) [61].

Eurycomanone is a cytotoxic bioactive ingredient found in E. longifolia Jack, that has a cytotoxic response against many epithelial cell types. The antiproliferative activity of eurycomanone was investigated on cancerous cell lines (Caov-3, HeLa, Hep G2, HM3KO and MCF-7) and it was found to be relatively nontoxic on noncancerous cell lines (MDBK, Vero). Eurycomanone proved to be cytotoxic towards HeLa cells by triggering apoptotic cell death [141].

Tong et al. investigated the in vitro and in vivo anti-cancer activities of a standardized quassinoid mixture (SQ40) from E. longifolia on LNCaP human prostate cancer cells, and showed that it induced selective cytotoxicity on human prostate cancer cells and inhibited the growth of LNCaP cells. SQ40 down-regulated the expression levels of G1-to-S phase transition regulatory proteins, cyclin D1, CDK4 and CDK2 and up-regulated cyclin inhibitor protein, p21Waf1/Cip1 which subsequently led to cell cycle arrest in G0/G1 phase. The anti-tumorigenic activity of SQ40 was successfully demonstrated in the mouse xenograft model [142].

Recently, Hajjouli et al. concluded that E. longifolia constituents, eurycomanone and eurycomanol are the regulators of signaling pathways involved in proliferation, cell death and inflammation. Both eurycomanone and eurycomanol inhibited Jurkat and K562 cell viability and proliferation without affecting healthy cells. Furthermore, eurycomanone inhibited NF-κB signaling pathway through inhibition of IκBα phosphorylation and upstream MAPK (mitogen activated protein kinase) signaling. Eurycomanone and eurycomanol present differential toxicity towards leukemia cells, and eurycomanone having the α,β-unsaturated ketone could be prerequisite for the NF-κB inhibition [143].

Wnt signaling regulates various processes such as cell proliferation, differentiation, and embryo development. 9-hydroxycanthin-6-one, decreased the expression of Wnt signal target genes, mitf and zic2a, through the activation of GSK3β independent of CK1α [144].

The quassinoids isolated from E. longifolia have been studied for thir in vitro cytotoxicities against KB cells derived from human epidermoid carcinoma of the nasopharynx [140]. Itokawa et al., isolated a new squalene-type triterpene, named eurylene, from E. longifolia which were found to be cytotoxic [53]. Chan et al., isolated a new C19 quassinoid 6α-hydroxyeurycomalactone from the roots of E. longifolia and have reported that the cytotoxic activity of these quassinoids was not mediated through DNA cleaving properties [49].

Chronic myelocytic leukemia (CML) is a malignant disease of the human hematopoietic stem cell which is characterized by a marked increase in granulocytes bone marrow hyperplasia and splenomegaly [145]. CML accounts for 15–20 percent of all leukemias [145,146] with a worldwide incidence of 1–2/100,000 [147,148,149]. The various isolates and purified eurycomane, an active compound from the roots of E. longifolia, were examined for their cytotoxic effect in K-562 cells isolated from patients with chronic myelocytic leukaemia (CML).

Al-Salahi et al., assessed the in vitro and in vivo anti-proliferative and apoptotic potentials of E. longifolia on K-562 leukemic cell line. Intraperitoneal administration of TAF273 (E. longifolia fraction, 50 mg/kg) resulted in a significant growth inhibition of subcutaneous tumor. TAF273 showed potent anti-proliferative activity in vitro and in vivo models of Chronic Myelogenous Leukemia (CML) and therefore, justifies further efforts to define more clearly the potential benefits of using TAF273 as a novel therapeutic strategy for CML management [150]. The cytotoxic activity of quassinoids was not found to be mediated through DNA cleaving properties [49]. In vitro, the anticancer effects of a fraction of E. longifolia were due to apoptosis via a caspase-9 and p53-independent manner [151] that perhaps involved Bcl-2 protein [152].

Angiogenesis, a process of formation of new branches of blood vessels, is strongly implicated in several important physiological situations [153,154]. Dysregulation of angiogenesis is involved in several pathological conditions, including atherosclerosis, proliferative retinopathies, rheumatoid arthritis, psoriasis, tumor growth and metastasis [155]. It is well recognized that angiogenesis is essential for the growth and metastasis of most solid malignancies, an increased body of evidence supports the enhancement of angiogenesis in hematologic malignancies as well [156]. Therefore, angiogenesis is currently becoming an important target for chemotherapeutic approaches in cancer therapy [157].

Antiangiogenic potential of partially purified quassinoid-rich fraction (TAF273) of E. longifolia root extract was evaluated using ex vivo and in vivo angiogenesis models and the anti-angiogenic efficacy of TAF273 were investigated in human umbilical vein endothelial cells (HUVEC). In vivo, it causes significant suppression in sprouting of microvessels in the rat aorta (IC50, 11.5 μg/mL), and shows a remarkable inhibition (63.13%) of neovascularization in chorioallantoic membrane of the chick embryo (IC50, 50 μg/mL). In vitro, TAF273 significantly inhibited the major angiogenesis steps such as proliferation, migration and differentiation of HUVECs. Thus, E. longifolia could be the potential source of promising therapeutic agents to treat angiogenesis-related disorders [158].

Fractions of E. longifolia extract have also been reported to induce apoptosis in breast cancer cells [152]. Further, Tee et al., elucidated the mode of action of F16 (a plant-derived pharmacologically active fraction) and observed that the intrinsic apoptotic pathway was invoked, with the reduction of Bcl-2 protein. It was concluded that the F16 from E. longifolia exerts anti-proliferative action and growth inhibition on MCF-7 cells through apoptosis induction, and that it may have anticancer properties [151].

The anti-proliferative, apoptotic and differentiating activities of partially purified sub-fractions (F1–F3) of E. longifolia root extracts were investigated on HL-60 leukemic cells. F1 showed unremarkable growth inhibition rate while F2 and F3 showed growth inhibitory effects with median inhibitory concentration (IC50) values of 15.2 and 28.6 µg/mL, respectively. E. longifolia extract (F2) showed promising anti-leukemic activity and can be a candidate for the development of a drug for the treatment of acute promyelocytic leukemia (APL) [159].

Nurhanan et al., evaluated the methanol, n-butanol, chloroform and water extracts obtained from the root of E. longifolia for its possible cytotoxic effect against KB, DU-145, RD, MCF-7, CaOV-3, and MDBK cell lines. Their results indicated that except for the water extract, all the other extracts produced significant cytotoxic effecte on these cell lines with no significant cytotoxic effect on MDBK (kidney) normal cell line. An alkaloid, 9-methoxycanthin-6-one was detected in each extract with different intensities, and was envisaged to be responsible for the observed activities [160].

Razak et al., reported that the extract of E. longifolia is found to be cytotoxic with IC50 of 11 μg/mL and 13 μg/mL on Hep2 and HFL1 cell lines respectively and that the combined extracts of E. longifolia and Hunteria zeylanica are more cytotoxic than the single extract on Hep2 cell lines [161].

5.4. Antimicrobial Effects
Farouk et al., showed that the alcoholic and acetone extracts of the leaves and stem were active on both the Gram-positive and Gram-negative bacteria Escherichia coli and Salmonella typhi. The root extracts had no antibacterial activity against the Gram-positive and Gram-negative bacteria tested. Aqueous leaves extract showed antibacterial activity against Staphylococcus aureus and Serratia marscesens [162].

Extracts from E. longifolia and L. pumila leaves were evaluated and analyzed for their antibacterial activity against human pathogenic Gram positive (Staphylococcus aureus) and Gram negative (Pseudomonas aeruginosa) bacteria. The extracts were prepared in different solvents (acetone, methanol, ethanol, and phosphate buffer) and at various concentrations ranging from 5 to 100 mg/mL. Most of the extracts showed relatively high antibacterial activity against the tested bacteria with inhibition zone diameters ranging between 7 and 25 mm. The minimum concentration of E. longifolia and L. pumila extracts which inhibited the growth of S. aureus and P. aeruginosa was 75 mg/mL in ethanol and 25 mg/mL in a phosphate buffer, respectively [163].

Kong et al. screened natural extracts from six plants, including E. longifolia, that improved the survival of S. aureus-infected worms by at least 2.8-fold, suggesting that these extracts could possibly activate host immunity to eliminate the bacteria or possibly interfere with the factor/s that prevent pathogen accumulation [164].

5.5. Anti-Inflammatory Effects
It was demonstrated that the β-carboline alkaloid 7-MCPA (7-methoxy-(9H-β-carbolin-1-yl)-(E)-1-propenoic acid) isolated from E. longifolia hairy-root cultures activated Nrf2 via a ROS-dependent p38 MAPK pathway and 7-MCPA anti-inflammatory effects was associated with 7-MCPA-induced activation of the Nrf2/HO-1 pathway. This study clarified the molecular mechanisms underlying the anti-inflammatory activities of β-carboline alkaloids of E. longifolia, which may be useful to prevent or treat inflammatory diseases [165].

Eurycomalactone, 14,15β-dihydroklaieanone, and 13,21-dehydroeurycomanone were identified as potent NF-κB inhibitors with IC50 values of <1 μM [45]. Varghese et al, studied hydroalcoholic extract of E. longifolia Jack for its antioxidant and in vitro anti-inflammatory properties. The antioxidant activity (free radical scavenging) was evaluated to determine the total antioxidant capacity of extract E. longifolia. The DPHH assay showed significant antioxidant activity in all concentrations used (i.e., 10, 25, 50, 100 and 250 µg/mL). The human RBC (HRBC) stabilization method was utilized to evaluate the in vitro anti-inflammatory activity of the extract, and it was found that this anti-inflammatory activity increased in a concentration dependent manner [82].

5.6. Anti-Anxiolytic Effect

The anti-anxiety effect of various fractions of E. longifolia was investigated in mice using various behavioral tests, including the open field (emotional state), elevated plus-maze (anxiolytic and anxiogenic drug effects), and anti-fighting test. The E. longifolia anxiolytic effect was similar to that of the positive control diazepam [166].

In human, effects of E. longifolia hot-water extract was screened for stress hormones and mood state in 63 subjects (32 men and 31 women) for moderate stress, with placebo for 4 weeks, and indicates that daily supplementation with E. longifolia extract improves stress hormone profile and certain mood state parameters [167].

5.7. Antidiabetic Effect

Blood glucose decreased in streptozotocin-induced hyperglycemic adult rats after treatment of 150 mg/kg body weight using aqueous extracts of E. longifolia. Blood-glucose levels decreased 38% (p < 0.05) and 47% (p < 0.001) for two different E. longifolia extracts. In normoglycaemic rats, no significant reduction was noted when the same extracts were used [168].

E. longifolia root extract increased insulin sensitivity through the enhancement of glucose uptake by more than 200% at 50 μg/mL and suppressed lipid accumulation in a concentration-dependent manner, suggesting the ability of E. longifolia to suppress lipid production would provide additional benefits in the treatment of diabetes [169].

5.8. Osteoporosis Preventive Effect

Osteoporosis in men is attracting more interest as it is becoming one of the main causes of morbidity and mortality in older men. Approximately 2 million men in the United States suffer from osteoporosis [170]. Worldwide, 1 in 3 women over 50 will experience osteoporotic fractures, as will 1 in 5 men [171,172,173]. According to Tambi and Kamarul, E. longifolia contains high concentrations of superoxide dismutase (SOD), an antioxidant that plays an important role in counteracting oxidative stress [120]. Other components of E. longifolia, such as alkaloids and triterpenes, can also act as antioxidants that may reduce bone loss and maintain the bone formation rate [123].

Recently, it was established that E. longifolia may be used in the prevention and treatment of osteoporosis, or more specifically, male osteoporosis. Shuid et al., showed that both testosterone replacement and E. longifolia supplementation to orchidectomised rats were able to maintain the bone calcium levels, with the former showing better effects, so E. longifolia prevented bone calcium loss in orchidectomised rats and therefore, has the potential to be used as an alternative treatment for androgen deficient osteoporosis [174].

The bioactive complex polypeptides from the E. longifolia root extract, labelled as eurypeptides, can exert and enhance their effects on the biosynthesis of various androgens [175]. Eurypeptides work by stimulating dihydroepiandosterone (DHEA). DHEA in turn will act on androgen receptors to initiate the conversion of androstenedione and androstenediol to testosterone and estrogen, respectively [125].

These eurypeptides may also alleviate SHBG and subsequently increase the free testosterone level [176]. Due to these proandrogen properties of E. longifolia, it is able to stimulate osteoblast proliferation and differentiation, resulting in increased bone formation rate. High levels of testosterone and estrogen may also exert proapoptotic effects on osteoclasts, reducing the bone resorptive activity. As testosterone levels decrease with age, it has been suggested that men can consume E. longifolia (at suitable dosages) as a supplement [177]. Other than its proandrogenic properties, E. longifolia contains high levels of nitric oxide (NO) [178] that have effects on bone.

Male osteoporosis can also be explained in terms of an oxidative stress mechanism. Free radicals, mainly reactive oxygen species (ROS), are efficiently scavenged in the body. However, oxidative stress will occur when there is an imbalance between increased ROS levels and inadequate antioxidant activity [179].

Orchidectomy (a model of androgen-deficient osteoporosis), can promote up-regulation of ROS which leads to oxidative stress. Oxidative stress plays a role in osteoblast apoptosis and osteoclast differentiation [180]. There are several mechanisms proposed for its antiosteoporotic effects.

The main mechanism is via its testosterone-enhancing effects for the prevention and treatment of androgen-deficient osteoporosis. Other mechanisms involved are through its nitric oxide generation and antioxidative properties. Due to E. longifolia’s safety profile and potential as an alternative antiosteoporotic agent, further studies are warranted to document a better and conclusive mechanism for its therapeutic action [123].

Androgen-deficient osteoporosis in men is treated with testosterone therapy, which is associated with many side effects. E. longifolia is known to possess androgenic properties and has been reported to protect bone from androgen-deficient osteoporosis in experimental animal models [181]. The combination therapy of E. longifolia and low-dose testosterone has potential for treatment of androgen-deficient osteoporosis.

The lower testosterone dose is beneficial in reducing the side effects of testosterone therapy [181]. E. longifolia exerts proandrogenic effects that enhance testosterone levels, as well as stimulate osteoblast proliferation and osteoclast apoptosis [123]. E. longifolia has been shown recently to protect against bone calcium loss in orchidectomised rats, the model for androgen-deficient osteoporosis.

Supplementation with it extract elevated the testosterone levels, reduced the bone resorption marker and upregulated OPG gene expression of the orchidectomised rats. These actions may be responsible for the protective effects of E. longifolia extract against bone resorption due to androgen deficiency [182].

Further studies on the regulation of OPG production by E. longifolia may provide insight into this novel mechanism. E. longifolia exerts proandrogenic effects that enhance the testosterone level, as well as stimulate osteoblast proliferation and osteoclast apoptosis. This will maintain bone remodelling activity and reduce bone loss. Phytochemical components of E. longifolia may also prevent osteoporosis via its antioxidative property. Hence, E. longifolia has the potential as a complementary treatment for male osteoporosis [123].

5.9. Miscellaneous Effects

5.9.1. Hormonal Effects

A standardized extract of E. longifolia Jack containing a high concentration of quassinoids (20% eurycomanone and 4% 13α,21-dihydroeurycomanone) had antiestrogenic effects against 17α-ethynylestradiol (EE)-induced uterotrophy of immature rats [86]. Another study showed that the E. longifolia plant extract normalized irregular estrous cycles and reduced the follicular morphological damage caused by chronic testosterone administration in the female rats. The reversal effect derived from the anti-estrogenic properties of the plant quassinoids. Further work is required to identify the exact mechanism behind the ameliorative effects of E. longifolia [183].

5.9.2. Ergogenic Effects

The ergogenic effects of E. longifolia were discussed in a review [184]. The authors reviewed its medicinal properties and studies investigating physiological responses and endurance exercise performance. Increased testosterone, as shown in animal models [115], has been suggested in anecdotal reports as being responsible for E. longifolia-induced increases in muscle mass and strength in humans. According to secondary sources, E. longifolia enhances testosterone production by the Leydig cells and frees bound testosterone for use by muscles [185].

5.9.3. Insecticidal Effects

E. longifolia-containing smoke from mosquito coils resulted in increased knock-down activities of mosquitos, but not increased mortality [186]. One study showed that E. longifolia exhibits the highest anti-protozoal activity at 1.0 mg/mL. The ethyl acetate fraction exhibited a slightly higher percentage of anti-protozoal activity and demonstrated the highest anti-protozoal activity against Blastocystis sp. isolates and showed a sizeable reduction in the cell count in comparison to the allopathic drugs [187].

5.9.4. Muscular Effects

In animal research, E. longifolia extracts increased weight of the levator ani muscle (involved in tail wagging) in castrated animals, but not testosterone-treated animals and uncastrated animals [188].

5.9.5. Antiulcer Effect

A bioassay study of Pasak bumi (E. longifolia) led to the isolation of four quassinoids, pasakbumin-A, -B, -C, and -D. Both pasakbumin-A (eurycomanone) and pasakbumin-B exhibited potent antiulcer activity [50]. In one other study, Qodriyah et al., investigation showed that E. longifolia in Radix is as effective as ranitidine in the treatment of ethanol-induced gastric lesions in rats [189].

5.9.6. Anti-Rheumatism

Effect Studies showed that decoction, and an alcoholic extract of E. longifolia roots are used to treat rheumatism [45,190].

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