DAIBIO Laboratory Research Ageless Man Toxicity in Jan 2024 with University and Academy

Authors:
Dr. Le Ngoc Hung^*1,2, Dr. Tran Van Thanh³, MSc. Nguyen Duc Anh ¹, MSc. Dai Thi Viet Lan⁴

1. Research and Technological Transfering Center - National Academy of Science in Vietnam
2. Science and Technology Academy - National Academy of Science in Vietnam
3. Vietnam University of Traditional Medicine
4. DAIBIO Traditional Medicine Company

This valued scientific work was published in National Journal of Analytical Sciences in Biology, Chemistry and Physic under Vietnam Ministry of Science and Technology in Jan 2024 as follows:
https://vjol.info.vn/index.php/TCPTHLS/article/view/99181

A new male wellness hard capsule Ageless Man (AM) was produced containing the extracts of Peliosanthes

micrantha (PME) 100mg, Cistanche tubulosa (CTE) 50mg, Cynomorium songaricum (CSE) 50mg,

Epimedium brevicornu (EBE) 40mg, Aѕtragaluѕ membranaceuѕ (AME) 68mg, Angelica sinensis (ASE)

68mg, Salvia miltiorrhiza (SME) 56mg, Paeoniae alba (PAE) 68mg. Before pharmacological study, its in

vivo toxicity studies were conducted. The results showed non-acute toxicity at dose of 17,5g/kgP and not yet

determined LD50; non-semi-chronic toxicity was at dose of 400 mg/kgP during consecutive 28 days.

Herbal Ingredients
The small-leaf stone lily root was collected in Krông Nô - Đắk Nông. Stem pieces of Cynomorium songaricum, rhizomes of Balanophora alata, leaves of Epimedium (Horny Goat Weed), roots of Salvia miltiorrhiza (Danshen), and roots of Paeonia lactiflora (White Peony) were purchased in Sapa - Lào Cai. Angelica sinensis roots were sourced from Đà Lạt – Lâm Đồng, and Astragalus membranaceus roots were bought from the Ninh Hiệp medicinal herb market in Hanoi.

Solvents, Experimental Animals, and Equipment
The extraction solvents used were food-grade 96° ethanol (Vietnam) and distilled water.
Experimental animals included Swiss albino mice (both sexes, 18–24 g) and Wistar rats (both sexes, 200–220 g), provided by the National Institute of Hygiene and Epidemiology.
Equipment included an ultrasonic extraction device (EnviTech, Vietnam), a low-pressure rotary evaporator R-210 (Buchi, Switzerland), an analytical balance AB104-S (Mettler Toledo, Finland), a hard capsule filling machine NJP-260 (ANS, Vietnam), standard animal housing at Daibio Traditional Medicine Company, specialized blunt-tipped needles from Japan for oral administration, small animal dissection kits, a CX33 microscope with a LP114000A camera system (Olympus, Japan), an AU480 biochemical analyzer (Beckman Coulter, USA), and other laboratory tools.
Preparation of Extracts with 50° Ethanol and Ageless Man Product Formulation
Herbal extracts were prepared following previously described methods [12]: powdered ingredients were placed in cloth bags and soaked in 50° ethanol. After soaking for 24 hours, the extract was filtered through filter paper and concentrated by rotary evaporation under low pressure. The dried extract, with moisture content below 15%, was stored in glass containers at room temperature.
Each capsule of Ageless Man No6 contained the following: 100 mg Peliosanthes micrantha extract, 50 mg Cynomorium songaricum, 50 mg Balanophora alata, 40 mg Epimedium, 68 mg Astragalus membranaceus, 68 mg Angelica sinensis, 56 mg Salvia miltiorrhiza, 68 mg Paeonia lactiflora, and excipients to reach 600 mg per capsule (size 0) [12].
Acute Toxicity Study of Ageless Man No6
The acute toxicity study was conducted according to OECD guidelines and Ministry of Health regulations [17-20]. The experiment used 60 Swiss albino mice (both sexes, healthy, 18–24 g) supplied by the National Institute of Hygiene and Epidemiology. Before the experiment, the mice were fasted for 12 hours but had free access to water. After 12 hours, they were randomly divided into groups of 10 mice each.
The test groups were administered Ageless Man No6 hard capsule powder at a volume of 0.2 mL/10 g body weight per dose, three times within 24 hours, with 3-hour intervals between doses. The doses were increased across six groups: 5.0 g/kg, 7.5 g/kg, 10.0 g/kg, 12.5 g/kg, 15.0 g/kg, and 17.5 g/kg to determine LD50. Mice were reintroduced to food 2 hours post-administration and given water as usual. Observations were conducted continuously for the first 4 hours, and mortality and other indicators were recorded over the first 72 hours, continuing for 7 days post-administration.
Subchronic Toxicity Study of Ageless Man No6
The subchronic toxicity study followed OECD and Ministry of Health guidelines [20, 21], using 32 Wistar rats (both sexes, healthy, 200–220 g) provided by the National Institute of Hygiene and Epidemiology. The rats were separated by sex and divided into four groups (n=8):

Control group: administered 1 mL 0.1% NaCMC solvent daily.
Test group 1: 135 mg of the preparation in 0.1% NaCMC/kg body weight.
Test group 2: 270 mg/kg.
Test group 3: 400 mg/kg.
These doses correspond to estimated human doses of 2, 4, and 6 capsules/day.

Body weight gain was calculated at specific time points using the formula:
TNiLj (%) = (mNiLj - mNiL0) / mNiL0 × 100, where:

Ni = time points (7, 14, and 28 days).
Lj = group (control, j=0; or test groups, j=1, 2, 3).
mNiLj = body weight at time Ni for group Lj.

Data Analysis:
Data were processed using Excel and presented as Mean ± SD (standard deviation). Statistical analysis employed t-tests to evaluate significant differences compared to the negative control, with *P<0.05 considered statistically significant.

References:

[1] Li H, Jiang H, Liu J. Traditional Chinese medical therapy for erectile dysfunction. Translational Andrology and Urology. 2017 Apr;6(2):192-198. doi: 10.21037/tau.2017.03.02. PMID: 28540226; PMCID: PMC5422677.
[2] Wang T, Chen C, Yang M, Deng B, Kirby GM, Zhang X. Cistanche tubulosa ethanol extract mediates rat sex hormone levels by inducing testicular steroidogenic enzymes. Pharmaceutical Biology. 2015;25:1-7.
[3] Liu W, Xin Z, Xin H, Yuan Y, Tian L, Guo Y. Effects of icariin on erectile function and expression of nitric oxide synthase isoforms in castrated rats. Asian Journal of Andrology. 2005;7(4):381-388.
[4] Wu ZQ, Chen DL, Lin FH, et al. Effect of bajijiasu isolated from Morinda officinalis F. C. How on sexual function in male mice and its antioxidant protection of human sperm. Journal of Ethnopharmacology. 2015;164:283-292.
[5] Hu WL, Hu LQ, Cheng B, et al. Effect of Angelica sinensis on NOS activity in penile tissues of rats after crushing injury to cavernous nerves. Zhonghua Nan Ke Xue. 2001;7:29-33.
[6] Zhu B, Zhang QL, Hua JW, Cheng WL, Qin LP. The traditional uses, phytochemistry, and pharmacology of Atractylodes macrocephala Koidz.: A review. Journal of Ethnopharmacology. 2018 Nov 15;226:143-167. doi: 10.1016/j.jep.2018.08.023. PMID: 30130541.
[7] Luo Q, Li Z, Huang X, et al. Lycium barbarum polysaccharides: Protective effects against heat-induced damage of rat testes and H₂O₂-induced DNA damage in mouse testicular cells and beneficial effects on sexual behavior and reproductive function of hemicastrated rats. Life Sciences. 2006;79:613-621.
[8] http://www.qihuang99.com/bbs
]9] Nguyễn Văn Dũng. Research on “Formulating Sâm Nhung Tán Dục Đơn herbal pills and evaluating their effectiveness in treating reduced sperm quantity and quality in patients at Da Nang Traditional Medicine Hospital.” Da Nang Department of Health, 2017–2018.
[10] Wang JS, Dai HH, Zhang KG, Cao KG, Deng S, Bao BH, Feng JL, Meng FC, Li HS, Wang B. Mechanism of Huoxue Tongluo Decoction in the treatment of erectile dysfunction caused by ischemic stroke based on network pharmacology. Chinese Herbal Medicine. 2021 Apr 30;13(3):351-358. doi: 10.1016/j.chmed.2021.04.016. PMID: 36118932; PMCID: PMC9476638.
[11] Ma K, Zhao F, Ye MY, Zhou K, Huang WJ, Zhao JF, Ma YF, Shou QY, Lv BD. Neuroprotective effect of Hongjing I granules on erectile dysfunction in a rat model of bilateral cavernous nerve injury. Biomedicine & Pharmacotherapy. 2020 Oct;130:110405. doi: 10.1016/j.biopha.2020.110405. PMID: 32679461.
[12] Lê Ngọc Hùng. Research report on “Developing a cultivation model for Peliosanthes micrantha medicinal plants to create health-protective products in Krông Nô District, Đắk Nông Province.” A collaborative project between the Science and Technology Institute (Contract No. 144/HĐ-VHL) and the Đắk Nông Provincial Government (Contract No. 41/HĐ-SKHCN), 2022–2024.