Impact of acute, oral ingestion of hypoxoside from African potato on hepatic and renal function tests in HIV infected patients on combination antiretroviral therapy

Abstract

Objectives:

African potato (hypoxis hemerocallidea) is used against HIV to enhance immune-function, although no studies have evaluated its use in HIV infected individuals on combination antiretroviral therapy. The study aimed to evaluate the acute effects of orally administered hypoxoside, a constituent of African potato, on the hepatic and renal function in HIV infected individuals on tenofovir disoproxil fumarate/ lamivudine/ efavirenz regimen.

Methods:

This was an open-label, two-period, fixed-sequence, pre-post test study, pilot design. Ethical approval was obtained from Medical Research Council of Zimbabwe (MRCZ A/2045) and Medicines Control Authority of Zimbabwe (MCAZ CT134/2016). Blood samples were collected before and after administration of African potato tablets. Tablets were administered orally once daily at 15mg/ kg hypoxoside for 10 days. Hepatic function tests (ALT, AST, ALP, GGT, albumin, total/ direct bilirubin); renal function tests (eGFR, blood urea nitrogen, creatinine), serum electrolytes (sodium, potassium, chloride) were assayed. STATA was used for statistical analysis.

Results:

Twenty-six participants were enrolled (85% female). Median age (range) was 43 (28–52) years. Most had overweight Body Mass Index (46%) and were married (54%). No statistical difference was noted during hypoxoside for AST/ ALT/ ALP/ GGT/ albumin/ bilirubin. There were no changes in creatinine/ eGFR/ electrolytes. A mean significant increase in total protein (p=0.04) and decrease in blood urea nitrogen (p=0.04) were noted.

Conclusion:

Short-term exposure to hypoxoside from African potato appeared safe and was not associated with clinically significant changes in hepatic, renal function tests/electrolytes. There is further need to evaluate extent of systemic exposure during long-term use in a larger population.

1.1. Introduction

The use of traditional herbal medicines for primary healthcare has become more popular than orthodox medicines especially in low resource countries (Kamboj, 2000). In Africa, 80% of the population uses traditional herbal medicine (Willcox & Bodeker, 2004) because traditional medicines are more affordable and are more easily accessible than conventional medicines (Maroyi, 2013). In most African countries, traditional medicines are organically farmed (Ozioma & Chinwe, 2019) and patients often believe they are safer because they are natural. The highest prevalence group using traditional medicine is among people living with HIV/AIDS (PLWHA) (Hughes et al., 2012). Combination antiretroviral therapy (cART) is efficacious in the management of HIV/ AIDS through suppression of viral replication (WHO, 2018). It is common for patients receiving cART not to inform their health provider that they are consuming herbs. Patient safety becomes an issue because of possible drug-herb interactions, which may compromise the patient’s health.

As of 21 June 2019, no studies were found which evaluated the safety of traditional medicines among PLWHA who were also receiving cART in Africa. Clinical studies in Thailand, Iran and South Africa have focused on the efficacy of herbal extracts on decreasing viral load, and/or increasing CD4+T cell count (Kusum et al., 2004; Paydary et al., 2012; Tshibangu et al., 2004). African potato (hypoxis hemerocallidea) is among the herbs commonly used for management of human immunodeficiency virus (HIV) and its related opportunistic infections. It is used mainly in Southern African countries (Owira & Ojewole, 2009). Chinsembu (2019) reported that Hypoxis spp. are rich in hypoxoside, β sitosterol, aglycone rooperol, tannins, sterols and sterolins. H. hemerocallidea extract is known to inhibit HIV-1 reverse transcriptase, IC₅₀ = 17.4 μg/ml (Chinsembu, 2019).

Herbal formulations of African potato are available commercially as standardized capsules, tablets, tonics and tinctures containing 300 – 500 mg hypoxis hemerocallidea or sterols/ sterolins (Ncube et al., 2013). Other topical formulations include powders, face/ night creams, nasal sprays, soaps, tissue oils, toner and exfoliators (Botle Buhle Brands, 2020). African potato is used as an immune stimulant, for treatment of wasting diseases, testicular tumours, diabetes mellitus, urinary infection, cardiac disease among other conditions (Ncube et al., 2013).

In vitro, four hypoxis species (Hypoxis hemerocallidea, Hypoxis colchicifolia, Hypoxis rigidula and Hypoxis acuminata) reported a lack of cytotoxic or genotoxic effects (Verschaeve et al., 2013). Infusion of Hypoxis hemerocallidea extracts in rats resulted in elevated plasma creatinine concentrations and a decrease in glomerular filtration rate. These results suggested that H. hemerocallidea extract may impair kidney function (Musabayane et al., 2005). Another study showed that African potato increased the bioavailability of indinavir in Sprague-Dawley rats (Havenga et al., 2018). A few human studies have examined the safety of African potato (Gwaza et al., 2013; Mogatle et al., 2008; Smit et al., 1995). Smit et al. (1995) conducted a Phase I clinical trial in cancer patients. The dosing range was 1200 – 3200 mg standardized Hypoxis plant extract per day divided into 3 doses.

The dosing range was due to considerable interpatient variations. There were no conclusive results on the clinical and biomedical toxicity of hypoxoside. Of the 24 patients, one experienced an episode of anxiety, nausea, vomiting, diarrhea, dyspnoea, and rigors which were possibly hypoxoside related (Smit et al., 1995). Drug interaction studies have found that African potato does not alter the pharmacokinetics of ritonavir boosted lopinavir (LPV/r) and efavirenz (Gwaza et al., 2013; Mogatle et al., 2008). These studies were conducted on healthy volunteers. Drewes and Khan (2004) have stated that the traditional use of African potato for a long time constitutes a form of ‘clinical trial’, “since evidence of toxicity would have led to its abandonment by traditional healers long ago”.

Although African potato has been used since historical times (Drewes & Khan, 2004), limited research has been done to determine its use among PLWHA who are also on cART. Topical preparations of African potato would be expected to be generally safe since there is low bioavailability. This study focused mainly on an oral dosage form, which has been demonstrated to be absorbed (Albrecht et al., 1995). The aim of the present study was to evaluate the acute effects of hypoxoside, a constituent of African potato on hepatic and renal function in HIV-infected individuals on a tenofovir disoproxil fumarate (TDF), lamivudine and efavirenz regimen. The objectives were to evaluate liver function before and after administration of hypoxoside tablets in patients on first-line antiretroviral therapy and to evaluate the electrolyte and renal laboratory tests in HIV infected participants before and after administration of hypoxoside from African potato.

1.2. Material and methods

1.2.1. Study population

Inclusion criteria were adults aged between 18 – 60 years who were infected by HIV/ AIDS. These participants were on a triple regimen of tenofovir disoproxil fumarate, lamivudine and efavirenz (Tenolam E). The regimen had to be started at least 6 months prior to entry to be eligible. Females had a negative pregnancy test at screening and at each clinic visit, were non-lactating and practiced birth control methods. Participants were included if they were hypoxoside and African potato naïve, willing to consume hypoxoside tablets and sign the informed consent form.

Exclusion criteria consisted of consuming alcohol and using tobacco during the study period. Participants with a recent history of illicit drug or alcohol use were ineligible for the study. Those with acute illness and a family history of cardiac complications were excluded. Participants who were on any herbal medicines were excluded. Participants were restricted from taking any medications other than the study medications without the prior approval of the study team.

1.2.2. Study design and procedures

This study was a single group, open-label, two-period, fixed sequence, pre-post test pilot study to evaluate the acute effects of hypoxoside, a constituent of African potato on hepatic and renal function in HIV infected adults on cART. The rationale for pilot studies is to provide preliminary findings, guiding the feasibility of the methods and procedures for larger studies (Thabane et al., 2010).

An approved advert was displayed at the Opportunistic infections (OI) clinic. In October 2018, participants who responded to the call for the study were screened to ensure that they met the eligibility criteria. They were given study identification numbers in chronological order. Eligible participants were enrolled after providing written informed consent. On enrollment, a baseline 5ml blood sample was collected and stored into red Clot Activator Vacuum tubes (Batch number 170520, Expiry date 04/2019) and immediately sent to the laboratory for analysis.

Participants were given hypoxoside tablets to be orally administered at 15 mg/kg hypoxoside as a single dose once a day in the morning. This is the dose found in literature (Gwaza et al., 2013; Mogatle et al., 2008). This first dose was administered on 14 October 2018 under directly observed treatment, short-course (DOTS). Standardized commercial tablets of the purified extract of hypoxoside were used. African potato tablets were manufactured by Herbal Solutions t/a Le Sel Brands Pvt Ltd in Harare, Zimbabwe (batch number:180321, expiry date:10/2020). They were supplied by Health and All Herbal Shop in Harare. Each scored tablet contained 320 mg of hypoxoside only and patients were asked to take the number of tablets most closely equivalent to the dosing of 15 mg/ kg/ day.

Participants consumed hypoxoside tablets from day 2 to day 11 as shown in Table A.1. The half-lives of African potato (20 hours for minor metabolites and 50 hours for major metabolites) (Albrecht et al., 1995), were used to determine the time to reach steady-state. Since this was the first time hypoxoside tablets were used in HIV infected people on cART, the minimum time to exposure was used. Adherence monitoring was carried out using pill counts, diaries, patient reminders and investigator’s reminders. After day 11, participants were counselled to discontinue the use of hypoxoside. The study was conducted between October – November 2018. The study design is represented in Table A.1 For the safety-monitoring plan, a physician and an investigator of record were monitoring the participants for adverse events and other tests.

1.2.3. Laboratory analysis

After obtaining serum, liver function tests were analysed using the Mindray BS 120 chemical analyser. The parameters measured were total protein, albumin, total bilirubin and direct bilirubin. The enzymes measured were aspartate aminotransferase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP) and gamma-glutamyl transpeptidase (GGT). The results were compared to the population reference ranges as shown in Table A.3.

The health status of the kidneys was assessed using the urea, creatinine and electrolyte tests. Levels of sodium (Na⁺), potassium (K⁺) and chloride (Cl⁻) ions in blood serum were measured using the 3 plus Humalyte ion-selective electrode (ISE) machine. Serum creatinine and urea were measured using the Mindray BS 120 machine. The levels of the analytes were compared to the reference ranges and these are shown in Table A.4. Glomerular filtration rate was estimated using the Cockcroft-Gault formula (Hoek et al., 2003) and adjusted for gender.

1.2.4. Determination of sample size

Pilot studies test the feasibility of methods and procedures to be used on a larger scale and guide the planning of a large-scale investigation. Pilot studies may, therefore, not require sample size calculations (Thabane et al., 2010). In this study, a convenience sample was used. For this pre-post study design, each participant acted as their own control, hence minimizing inter-subject variability. As a result, sample size was smaller.

1.2.5. Statistical analysis

Descriptive statistics were used to describe the demographic characteristics of participants. Paired t-tests were used at 95% confidence interval (CI). A P value of <0.05 was considered to be statistically significant. All data analysis was performed using STATA® version 13.0

1.2.6. Ethical considerations

Site approval was obtained from the Joint Research Ethics Committee for The University of Zimbabwe College of Health Sciences and The Parirenyatwa Group of Hospitals (JREC 247/15) before initiation of study procedures. Ethics and IRB approval were obtained from the Medical Research Council of Zimbabwe (MRCZ A/2045) and the Medicines Control Authority of Zimbabwe (MCAZ CT134/2016). A signed and dated informed consent was obtained from the participants before any study-related procedures were performed. The study was conducted in accordance with the approved protocol, pertinent requirements of the Committee on Human Research ethics guidelines, and the Declaration of Helsinki (Association, 2001). The clinical trial was retrospectively registered on The Pan African Clinical Trials Registry (PACTR), registration number PACTR201909691101062.

1.3. Results

1.3.1. Demographics

Thirty-one participants presented to the clinic for screening. Three did not meet the eligibility criteria because of the following reasons: positive pregnancy test result, not on the required cART regimen and the presence of co-morbidities. Two other participants were excluded from data analysis because they did not complete all stages of the study. Twenty-six participants completed all phases of the study and they are described in Table A.2

1.3.2. Liver function tests.

As shown in Table A.3, most participants had normal results for the liver function tests performed. The Division of AIDS (DAIDS) table was used for grading and recording the severity of adult adverse events (DAIDS Table, 2017).

1.4. Discussion

This was the first study to evaluate the acute effects of hypoxoside on hepatic and renal function in HIV-infected individuals on a TDF, lamivudine and efavirenz regimen. The majority of the participants (85%) were women, and these findings are consistent with previous studies, which reported less participation of males in HIV research (Mantell et al., 2019). Generally, less males present to the National ART Programme than females in this setting (Mutasa-Apollo et al., 2014). There is a need to find ways to increase male participation in HIV/ AIDS research studies.

African potato has been used since the 1960s (Drewes & Khan, 2004), yet few clinical safety studies have been conducted on the plant. Results from rat models have suggested that African potato extracts may impair renal function. Male rats were infused with an aqueous extract of African potato over 1.5 hours (acute effects) and over 5 weeks (chronic effects). Chronic treatment reduced urinary Na⁺ output and reduced GFR (glomerular filtration rate) compared to controls. Plasma creatinine concentration was increased (Musabayane et al., 2005). In another study, an aqueous solution of African potato was administered to male rats orally at 200mg/ kg or 800mg/ kg. At lower concentrations, liver injury was ameliorated. Higher concentrations increased relative kidney weights and resulted in an elevated concentration of serum creatinine which could be due to reduced tubular secretion of creatinine, (Oguntibeju Oluwafemi O et al., 2016) confirming the study by Musabayana et al. (2005).

All clinical trials conducted on African potato or its phytosterols have shown it to be safe without adverse effects (Albrecht et al., 1995; Donald et al., 1997; Gwaza et al., 2013; Mogatle et al., 2008; Smit et al., 1995). The study by Smit et al. (1995) assessed the short to long-term toxicity of hypoxoside in 24 patients with lung cancer. The patients took between 1200 – 3200 mg standardized Hypoxis plant extract per day, up to 5 years. The values for ALT, AST and lactate dehydrogenase (LD) remained within normal limits (<60 U/ L, <40 U/L and ~140–300 U/L respectively). Serum electrolytes (Na⁺, K⁺, CI⁻, Ca²⁺) and serum creatinine and urea concentrations were mostly within normal limits. One of the patients who survived the longest later died of tuberculosis (TB) pneumonia after 5 years on hypoxoside therapy (approximately 1 g daily). This patient had normal ALP levels (~30–80 U/L) and an autopsy showed that all organs (kidney, liver, bone marrow, colon, intestine, brain, spleen) were normal. The conclusion was short- and long-term therapy with high hypoxoside dosages did not result in any obvious toxic effect among cancer patients (Smit et al., 1995).

A study by Mogatle et al., investigated whether the concurrent administration of a traditionally prepared African potato decoction would affect the pharmacokinetics of efavirenz in 10 healthy volunteers. The decoction was dosed at 15 mg/kg/day of hypoxoside administered over 14 days. Laboratory tests for liver function and urinalysis were normal and no serious adverse effects were reported for the study (Mogatle et al., 2008). Another study investigated the effect of the African potato capsules on the pharmacokinetics of ritonavir-boosted lopinavir (LPV/r). African potato was administered at 15 mg/kg/day of hypoxoside for 7 days. There were no clinically significant changes in laboratory results during this study (Gwaza et al., 2013).

For this study, no statistical difference was noted during the use of hypoxoside tablets for AST, ALT, ALP, GGT, albumin and bilirubin. Although not clinically significant, there was a mean increase in total protein from 75 U/ L ± 7.6 to 78 U/ L ± 6 (p=0.04). Proteins synthesized in the liver include albumin, clotting factors, immunoglobulins and carrier proteins. Factors including nutritional status, losses in the urine, hormonal factors, catabolism will affect albumin. Hepatic tests reflect different functions of the liver. They reflect metabolism in the liver or hepatocellular integrity (AST and ALT), formation and the subsequent free flow of bile (bilirubin and ALP), and protein synthesis (albumin). Raised values commonly indicate liver disease although some enzymes may be elevated by other conditions (Limdi, 2003).

Both AST and ALT are excellent markers of hepatocellular injury. ALT is more specific to the liver while AST is present in the liver and other tissues like cardiac muscle, skeletal muscle, kidneys, brain, pancreas, lungs, leucocytes, and red cells (Pratt & Kaplan, 2000). One female participant had elevated levels of AST (56.4 U/L), ALT (58.6 U/L), ALP (170 U/L), total protein (91.7 U/L) and albumin (73.5 g/L) after consuming hypoxoside. All values went back to normal 2 weeks after discontinuation. Common causes for raised AST/ ALT levels include non-alcoholic fatty liver disease, alcoholic liver disease, medications (antibiotics, herbs, NSAIDs, illicit drugs) chronic hepatitis B and C, autoimmune liver disease, haemochromatosis, strenuous exercise and coeliac disease (Limdi, 2003). Literature reports that a GGT level of twice the normal with an AST/ALT ratio of 2:1 or more is highly suggestive of alcohol abuse (Cohen & Kaplan, 1979). Thus for this participant, alcohol abuse was ruled out. This patient was asymptomatic and had normal values at baseline. After consuming hypoxoside all values were less than twice the normal value, hence they were of no clinical importance (Pratt & Kaplan, 2000).

Conventionally, the main markers used to measure kidney function are serum creatinine and blood urea nitrogen. eGFR is estimated using the clearance of creatinine and serum creatinine levels generally have an inverse relationship to eGFR. A rise in serum creatinine is associated with a parallel decrease in GFR which may imply a reduction in kidney function (Bagshaw & Gibney, 2008). Serum creatinine can be increased by a high protein diet, muscle mass, rhabdomyolysis, drugs (e.g. trimethoprim, cimetidine, spironolactone, probenecid). For this study, there were no changes in electrolyte (sodium, potassium, chloride) concentrations or renal function tests (creatinine and eGFR). Although it was not statistically significant (p=0.1), most participants (58%) had an eGFR below 80 at baseline. In PLHWA, the combination of HIV associated nephropathy (HIVAN) and TDF nephrotoxicity may worsen kidney damage (Mtisi et al., 2019).

There were no serious adverse events observed in this study. The reported side effects were increased appetite, increased libido, nausea and headache. The increase in appetite could explain the increase in total protein observed. Increased libido could be psychological because there are no aphrodiasics which have been screened in African potato. Nausea, anxiety, vomiting, diarrhea, dyspnea and rigors have been experienced by one patient in another study. This patient had serum concentrations of hypoxoside metabolites at 163 μg/ml (Smit et al., 1995).

Since this was an acute pilot study, the main limitation was the small sample size used and short duration. Pilot studies test the feasibility of methods and procedures to be used on a larger scale (Thabane et al., 2010). These preliminary findings have shown that hypoxoside tablets are generally safe on the liver and kidney during acute exposure. Hence, it is recommended that these effects should be evaluated in a larger sample size over a longer period in a crossover design with randomization. In addition, work can be conducted to evaluate a dose-response curve. Further studies can also be done to find out which fraction of protein is increased when patients consume African potato since this may be the one responsible for enhancing immunity.

1.5. Conclusions

Short-term exposure to hypoxoside tablets from African potato during cART appeared safe and was not associated with clinically significant changes in hepatic and renal function tests or electrolytes. There is a further need to evaluate the extent of systemic exposure to African Potato and its effects during long-term use in a larger sample size. Further studies can be carried out to evaluate the fraction of protein that is increased when African potato and it’s constituents are consumed plus the evaluation of a dose response curve for hypoxoside in African potato.

Highlights.

Short-term exposure to hypoxosidetablets during combination antiretroviral therapy:

• appeared safe in people living with HIV/ AIDS (PLWHA)

• was not associated with clinically significant changes in hepatic tests;

  • renal function tests or electrolytes.
• more research areas from this study include:

  • to investigate the extent of systemic exposure during long-term use in a larger trials
  • to evaluate the fraction of protein that is increased when African potato and it’s constituents are consumed
  • to evaluate a dose-response curve for hypoxoside in African potato

• It is feasible to conduct larger trials of the safety of African potato and its constituents in PLWHA.

Table A.1.

Summary of Study Design.

Baseline Data Blood sample	Tenolam-E plus African potato DOTS	Steady-state African Potato Blood sample
Tenolam-E alone	Tenolam-E plus African potato
Day 1	Day 2	Day 11

Table A.2.

Demographic Characteristics of Study Patients at Baseline. (n = 26)

Characteristic	Value (n = 26)
Sex, female, n (%)	22 (85)
Age, years, median (range)	43 (28 – 52)
BMI, n (%)
Underweight (<18.5)	1 (4)
Normal (18.5 – 24.9)	10 (38)
Overweight (25 – 29.9)	12 (46)
Obese (30 – 35)	3 (12)
Marital status, n (%)
Married	14 (54)
Widowed	8 (31)
Separated/ divorced	4 (15)
Religion, n (%)
Non-Apostolic Christian	21 (81)
Catholic	2 (7)
Apostolic Christian	1 (4)
Islam	1 (4)
Traditional	1 (4)
Efavirenz 400mg dose [vs 600mg], n (%)	22 (85)

Table A.3.

Results of Liver Function Tests (n = 26).

Parameter (normal reference range)	Before hypoxoside tablets, mean ± sd [95% CI]	After hypoxoside tablets, mean ± sd [95% CI]	p value*
•AST (<42 U/ L)	27.8 ± 5.8 [25.3 – 30.2]	29.0 ± 7.8 [25.7 – 32.2]	0.84
ALT (< 42 U/ L)	26.9 ± 7.6 [23.8 – 30.0]	23.9 ± 13.1 [18.6 – 29.2]	0.15
ALP (20 – 130 U/L)	109.8 ± 52.4 [88.6 – 131.0]	84.8 ± 35.9 [70.3 – 99.3]	0.09
Total protein (60 – 81 U/ L)	74.5 ± 7.6 [71.5 – 77.6]	77.6 ± 6.0 [75.2 – 80.0]	0.04
Albumin (35 – 50 g/ L)	42.9 ± 5.7 [40.5 – 45.2]	44.3 ± 6.8 [41.5 – 47.0]	0.40
GGT (15 – 85 U/ L)	39.3 ± 17.5 [32.2 – 46.3]	40.9 ± 14.3 [35.1 – 46.6]	0.62
Total bilirubin (3 – 21 μmol/ L)	6.5 ± 3.6 [5.0 – 8.0]	6.8 ± 2.5 [5.8 – 7.8]	0.75
Direct (conjugated) bilirubin (2.1 – 5.1 μmol/ L)	3.2 ± 1.1 [2.7 – 3.6]	3.2± 1.1 [2.7 – 3.6]	0.96

^•Outliers at baseline excluded from analysis (n=2)

^*p-value obtained from paired t-test

sd – standard deviation

CI – confidence interval

Table A.4.

Results of Electrolytes and Kidney Function Tests (n = 26).

Parameter (normal reference range)	Before hypoxoside tablets, mean ± sd [95% CI]	After hypoxoside tablets, mean ± sd [95% CI]	p value*
Sodium (135 – 150 mmol/ L)	143.0 ± 3.2 [141.7 – 144.3]	143 ± 4.5 [141.2 – 144.8]	0.96
Potassium (3.5 – 5.5 mmol/ L)	4.5 ± 0.4 [4.4 – 4.7]	4.5 ± 0.4 [4.3 – 4.6]	0.64
Chloride (85 – 110 mmol/ L)	107.8 ± 2.2 [106.9 – 108.7]	107.4 ± 2.2 [106.5 – 108.3]	0.47
Creatinine (55 – 145 μmol/ L)	93.7 ± 19.7 [85.8 – 101.7]	85.3 ± 12.0 [80.4 – 90.1]	0.07
eGFR (80 – 120 mL/ min/ 1.73m2)	77.4 ± 19.8 [69.4 – 85.4]	84.2 ± 20.7 [75.9 – 92.6]	0.10
Blood urea nitrogen (1.41 – 3.53 mmol/ L)	2.2 ± 0.6 [2.0 – 2.5]	1.9 ± 0.5 [1.7 – 2.1]	0.035

^*p-value obtained from paired t-test

CI – confidence interval

eGFR – estimated glomerular filtration rate

sd – standard deviation