Anticancer Activity of the Thai Herbal Formula “Kerra” Versus

Afatinib and Doxorubicin In Vitro

CHATCHAYA NANGSUE1,*, PUSSADEE SRATHONG2, KIATTAWEE CHOOWONGKOMON3,

KEERATIKORN PROMNIKORN3, CHAINARONG TOCHARUS4

1Faculty of Medicine,

Chiang Mai University,

110 Intawaroros Road, Suthep, Mueang Chiang Mai District, Chiang Mai 50200,

THAILAND

2Prachomklao College of Nursing,

Phetchaburi 7600,

THAILAND

3Department of Biochemistry, Faculty of Science,

Kasetsart University, Bangkok 10900,

THAILAND

4Department of Anatomy, Faculty of Medicine,

Chiang Mai University, Chiang Mai 50200,

THAILAND

*Corresponding Author

Abstract: - One of the greatest challenges impeding the achievement of global health goals and afflicting

millions of people each year is cancer. Prevailing cancer treatment options come with inherent risks, and

significant adverse effects, and can result in financial burdens. Nowadays, many turn to alternative medicine,

which have played an integral part in the treatment of a myriad of diseases especially when other approaches

are ineffective or unable to be deployed. However, an opulent number of herbs seldom have reliable evidence

derived from research to support their claims of phytochemical properties contributing to the alleviation of

illnesses. Kerra is a Thai herbal formula that possesses immunomodulatory properties and anti-tumor activities

which provides a safe, efficacious, and holistic treatment for cancer. Kerra offers therapeutic benefits without

resulting in severe adverse effects, ameliorates overall welfare and quality of life for advanced care patients,

and is cost-effective, allowing them to be vastly accessible for widespread use, particularly in areas facing

disparities due to insufficient economic stability. This study explores the in vitro anticancer effect of the Kerra

formula utilizing cytotoxicity assays on six cancer cell lines, compared to afatinib, a targeted therapy for non-

small cell lung carcinoma, and doxorubicin, a chemotherapeutic agent. Anticancer activity was determined by

3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay and the half-maximal inhibitory

concentrations (IC50) were calculated for the A431, A549, HepG2, HCT-116, NCI-H1975, MCF-7, and HeLa

cell lines. Kerra showed the best effectiveness against HCT-116 and A431. The IC50 values showing

inhibition of the growth of colorectal carcinoma (HCT-116 cell line) by Kerra, afatinib, and doxorubicin are

72.96, 3.574, and 63.39 µg/ml, respectively. The IC50 values for inhibition of epidermoid carcinoma (A431

cell line) by Kerra and afatinib are 96.1 and 2.449 µg/ml, respectively. The findings revealed the Kerra extract

can inhibit the growth of different types of cancer cells. From our results, Kerra may be advantageous to use in

conjunction with conventional cancer interventions, offer an economical solution, and improve the quality of

life remarkably for end-stage cancer patients by mitigating severe adverse events.

Key-Words: - Thai herb, Anticancer, Cytotoxic, Cell culture, Phytochemistry, Traditional medicine, Cancer cell

Received: August 5, 2023. Revised: December 23, 2023. Accepted: February 2, 2024. Published: April 1, 2024.

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1 Introduction

In today’s time, modern medicine has alleviated the

quality of life of people around the globe and has

provided accessible healthcare services. Research

efforts have yielded a plethora of medical

breakthroughs which have presented innovative

treatments and vaccinations for diseases that in the

past may have had a worse prognosis, leading to

morbidity and fatal illness. Nevertheless, cancer

remains an adversity where the key to optimum

interventions is still unknown, and is the second

leading cause of death worldwide accounting for 9.3

million deaths annually, [1] and the trend is still

unfortunately increasing. The global cancer burden

is predicted to escalate to 28.4 million cases in

2040, [2], instigating a threat to achieving global

health goals and welfare amelioration.

Devastatingly, the incidence of early-onset cancers

which includes breast, tracheal, prostate cancer, etc.

has risen by 79% from 1990 to 2019, [3].

Therefore, this exacerbates the impending need

to enforce primary prevention, awareness of risks,

screening, and search for efficient and accessible

treatment approaches to deter repercussions from

the burden of cancer. The latest medical practices

fail to overcome many challenges despite research

endeavors including high expenses and adversities

associated with treatment. A new approach is

needed to help modern medicine, the answer is

herbal medicine. Thai herbs have been used since

ancient times to ameliorate illness and symptoms.

Especially since the COVID-19 pandemic paved the

way for the common use of herbs to alleviate

illnesses ranging from mild to severe cases, in

prevailing times, an abundance of people turn to

ancient herbal remedies that are deemed to be safe

and are appealing because their prices are within

reach for frequent utilization.

A study conducted in a Thai Buddhist temple

herbal medicine hospice included cancer patients

who received an herbal remedy for cancer treatment

from the hospice and reported a statistically

significant improvement in symptoms such as pain,

fatigue, and insomnia which correlates with a better

quality of life, [4].

However, in Thailand, some traditional herbal

medicines are allowed to be placed on the market

without official registration as a drug, and people

still take these herbs albeit they may not have

enough conclusive evidence to prove their efficacy

and safety profiles. Conversely, Kerra is a Thai

traditional herbal formulation that proved to be

efficacious in tackling many diseases through a

holistic approach, for people of all ages, including

chronic inflammatory conditions, and has had

extensive research conducted to discover the

mechanism of action behind the medical properties

of the phytochemical compounds found in the

formula. It originally surfaced to widespread use

during the unprecedented crisis brought upon by the

COVID-19 pandemic as hospitals filled to the brim

with severe cases, so the redundant patients were

denied hospitalization. Many desperately searched

for treatment options and turned to traditional herbs,

Kerra, in particular during self-isolation. Self-

isolated SARS-CoV-2 patients taking Kerra

capsules had no increase in the severity of illness

with better treatment outcomes.

In addition to evidence shown from self-use by

the general population, Kerra’s mechanisms for

stopping replication of the virus are known. It can

inhibit inflammatory response and viral replication

enzymes in SARS-CoV-2. The medicinal properties

in the herbs that constitute Kerra for example are

relief of fever, suppression of inflammation, and

stimulation of macrophages then boosting the

immune system contributing to its holistic effects.

Furthermore, research shows that Kerra

possesses anti-inflammatory properties,

immunomodulatory actions, and anti-tumor

activities. The Kerra extract can reduce

inflammation by suppressing IL-6, activate

apoptosis and cell death in colorectal cancer cell

lines, suppress cell proliferation of adenocarcinoma

cell lines via the EIF2 signaling pathway, and may

induce apoptosis in HCT116 cells through the

regulation of caspase-8 and caspase-9, [5].

The Takkasila Scripture is what Kerra medicine

is derived from. It has medical expertise in treating

epidemics and fevers and is a highly valuable

resource for Kerra. The Takkasila scripture is 1 of

14 scriptures in the Thai royal textbook of

traditional medicine. Kerra has been used to treat

patients with chronic diseases who have been

through countless medical interventions with no

hope left. It has helped patients with infectious

diseases such as COVID-19, & influenza, cancer,

autoimmune and inflammatory conditions such as

rheumatoid arthritis, psoriasis, systemic lupus

erythematosus, inflammatory bowel disease,

irritable bowel syndrome, and patients with diabetes

and its complications. Currently, there are limited

treatment options for many of these conditions also

entailing severe side effects and fees. Tinospora

crispa (L.) Hook. f. & Thomson, an extract found in

Kerra was found to be insulinotropic in cell culture

and animal studies, so it can be inferred that it can

be a potential drug for diabetes mellitus, [6].

An investigation of some Thai herbal medicine

extracts on inhibition of cholangiocarcinoma cell

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lines proved to be successful, [7].

Cholangiocarcinoma has a high incidence,

especially in Northeastern Thailand and is

aggressive. This shows that phytochemicals in herbs

show potential benefits for investment in cancer

research as potent drug options. Proteomics studies

on Kerra analyzed its apoptotic pathways in

colorectal cancer cell lines and revealed dose-

dependent inhibition, instigation of apoptotic

activity, and regulation of key proteins involved in

cell death and proliferation pathways such as MYC

and CDKN1A, [5]. There is sufficiently strong

evidence that Kerra delivers an efficacious measure

to tackle a diverse amalgamation of diseases, most

significantly cancer, without severe side effects,

alleviates quality of life, especially in advanced care

patients, and is inexpensive, which can become

extremely accessible for use on a widescale to

pertain to the scarcity of vital resources. Kerra has

11 bioactive constituents as shown in Table 1.

Table 1. List of herbal extracts in the Kerra

formulation

2 Materials and Methods

2.1 Compound Preparation

The Kerra powder from the capsules was dissolved

with 100% DMSO to reach 100 mg/mL, then 2-fold

serial dilution was used to get 9 concentrations. The

freshly prepared solution was diluted to the final

concentrations of 1250, 625, 312.5, 156.25, 78.125,

39.06, 19.53, 9.77, and 4.88 µg/mL in the culture

medium with a DMSO concentration of no more

than 1% (v/v).

2.2 Cell Culture

The A431, A549, HepG2, HCT-116, NCI-H1975,

MCF-7, and HeLa cell lines were acquired from The

American Type Culture Collection (ATCC). The

A431, A549, MCF-7, and HeLa cells were cultured

in the Dulbecco's modified Eagle's medium

(DMEM). The HepG2 cells were cultured in the

Eagle's Minimum Essential Medium. The HCT-116

cells were cultured in McCoy's 5A Medium. The

NCI-H1975 cells were cultured in the Roswell Park

Memorial Institute (RPMI) 1640 Medium. All cell

types included 10% fetal bovine serum (FBS) and

1% penicillin/streptomycin. The incubator

conditions for cell culture were a humidified

atmosphere of 5% CO2 at a temperature of 37°C.

The respective media and cell density (cells/well) of

each cell line are shown in Table 2.

Table 2. Cell type, media, and cell density

(cells/well) of cancer cell lines

2.3 Cytotoxicity Assay

The MTT test was used to determine cancer cell

growth inhibition. The change of a tetrazolium dye

to an insoluble formazan compound by viable cells

was measured. Cancer cell lines were placed in 96-

well plates at a density of 6 × 103 cells per well

except for HepG2 which was 15 × 103 cells per well.

The plates were incubated overnight at 37 °C with

5% CO2. Then the cells were treated with 100 μL of

varying concentrations of the Kerra in ethanol

solution and incubated for 72 hours. Anticancer

drugs, Afatinib and Lapatinib (Santa Cruz

Biotechnology, USA) were the reference

compounds. The culture medium was changed to

100 μL of 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl

tetrazolium bromide (MTT) (Thermo Fisher

Scientific, USA) solution (0.5 mg MTT in 1 mL

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culture medium) and incubated at 37 °C for 3 hours.

After that, the medium was discarded and 50 μL of

DMSO was placed in each well for solubilization of

formazan. The absorbance of each well was

measured at 570 nm using a microplate

spectrophotometer (Synergy HTX Multi-Mode

reader, BioTek, USA). The half-maximal inhibitory

concentration (IC50) values were determined using

the absorbance of Kerra-treated wells to the

untreated control. All experiments were done in

triplicate.

2.4 Statistical Analysis

The IC50 values and coefficient of determination

(R2) were calculated using the concentration of

Kerra and the respective percent inhibition of each

cell line from triplicate experiments using GraphPad

Prism Software Version 10.0.0.

3 Results

3.1 Anticancer Activity on Cell Lines

Table 3. Cancer cytotoxic effects comparison in

different cell lines

The in vitro investigation reveals that Kerra is

efficacious at inhibiting the proliferation of cancer

cells shown by its toxic impact on the A431, A549,

HepG2, HCT-116, NCI-H1975, MCF-7, and HeLa

cell lines, as shown in Table 3. Its low IC50 values,

lack of discrimination against cell types, and dose-

dependent response support Kerra as a potential

treatment method. Comparisons to afatinib and

doxorubicin show that Kerra has effective

anticancer activities and benefits as a natural

phytochemical that plays a role as an alternative to

conventional treatment. The experiment was not

conducted for the MCF-7 cell line for afatinib, and

the A431 and NCI-H1975 cell lines for doxorubicin.

The dose-dependent inhibition activity of the Kerra

solution with a 0.5% DMSO concentration on all the

cell lines is shown in Figure 1, Figure 2, Figure 3,

Figure 4, Figure 5, Figure 6 and Figure 7. The IC50

is represented as the mean SD of triplicate

experiments.

Fig. 1: Kerra on A431 cell line growth inhibition

Fig. 2: Kerra on HCT116 cell line growth inhibition

Fig. 3: Kerra on HepG2 cell line growth inhibition

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Fig. 4: Kerra on MCF-7 cell line growth inhibition

Fig. 5: Kerra on A549 cell line growth inhibition

Fig. 6: Kerra on NCI-H1975 cell line growth

inhibition

Fig. 7: Kerra on HeLa cell line growth inhibition

The IC50 curves show that Kerra is effective at

inhibiting the proliferation of cancer cells.

Comparing between cell lines, we can see that Kerra

does not discriminate inhibition between each

cancer type.

4 Discussion

This study’s findings open a new door towards a

new way of cancer treatment, without detrimental

repercussions, highlighting the anti-tumor potency

of Kerra through an in vitro approach. Kerra may be

comparable to conventional therapeutic agents in

terms of its ability to kill a diverse range of cancer

types. The herbal formula has identified bioactive

compounds that contribute to its anti-inflammatory

effects, immunomodulatory actions, and anti-tumor

activities that are all beneficial to cancer cell growth

inhibition. Kerra is 500mg per capsule, and many

capsules can be taken orally per day along with

other herbal formulas to fight cancer, which is more

convenient than chemotherapy sessions requiring

hospitalization.

A cohort investigating the use of traditional

Chinese medicine in gastric cancer patients after

receiving surgery reported a statistically significant

decrease in deaths compared to patients who did not

use herbs and they concluded that the medicine may

be possible to use in gastric cancer patients after

surgery and adjuvant chemotherapy, [8]. This

enhances the strength of support behind

phytochemicals as a successful measure against

diverse diseases. Kerra has also been taken in self-

isolated SARS-CoV-2 patients and showed

improved treatment outcomes not requiring

hospitalization as a result of the herbs’ anti-

inflammatory effects which can benefit cancer

treatment via modulation of tumor environment.

Adversely, some safety profiles remain unknown

when it comes to using herbs in conjunction with

conventional cancer therapy. A review reported

potential anticancer drug-herb interactions with

herbs from the 2020 Thai Herbal Pharmacopoeia,

[9]. Clinical studies on Kerra are still ongoing so its

efficacy and safety may be available in the future

and benefit modern cancer treatment, but current

preclinical studies and general use in the population

have not brought about any adverse events and have

drastically improved the well-being of stage IV

cancer patients due to the synergistic effects of each

herbal ingredient in the formula.

Kerra is derived from ancient medicinal

practices that focuses on a holistic approach to allow

the body to maintain in a condition which the

immune system can act effectively and be protected

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from impairment, to fight against diseases or

pathogenic agents. In addition, ancient medicine

from a myriad of herbal extracts calls for synergistic

effects from the combination of its bioactive

constituents, increasing potency and lowering

adverse effects to a minimum. Modern medicine is

based on a different mechanism, using a single

chemical compound to inhibit or agonize a specific

target, which inevitably leads to severe side effects

impacting well-being.

An oral pill form targeted therapy that

irreversibly inhibits the ErbB family of tyrosine

kinases for non-small cell lung cancer with non-

resistant epidermal growth factor receptor (EGFR)

mutations is Afatinib, [10]. Since it is a targeted

therapy, fewer side effects occur, such as diarrhea,

acneiform eruptions, and stomatitis, but may vary in

severity, [11], [12]. Doxorubicin is an anthracycline

chemotherapeutic agent used in a variety of

different cancer types but can bring along many

systemic side effects especially cardiotoxicity, [13],

[14], [15]. Current therapeutic options are effective

but seldom come without drastic adverse events, so

herbs with anti-tumor activities like Kerra should be

further investigated as an alternative that can lead to

potential medical breakthroughs in cancer treatment.

Moreover, in Thailand, incidences of

cholangiocarcinoma, an aggressive type of cancer,

are highly prevalent in the north-eastern region.

Pollution from fine particulate matter that is 2.5

microns or less in diameter (PM2. 5) has exceeded

the standards of safety for many years and has been

correlated to an increase in the incidence of lung

cancer. With the rise in the trend of cancer and a

known cancer distribution pattern within the

population, the discovery of novel interventions is

warranted, and the key may be herbal drugs like

Kerra.

Prevailing traditional medicine usage and

ethnomedicine are not strictly monitored, leading to

false information, dangerous adversities, and

unsuccessful treatment. However, if competent

herbs with strong evidence from research can be

pushed for legal registration and commercialization,

more people can look for affordable, safe, and

effective treatment. Further research is warranted to

investigate the efficacy and safety profiles of the

herbs in larger clinical studies and its potential to

become an effective and economical cancer cure

with improved quality of life for frail patients in the

real world. Kerra has high potential and supporting

evidence for utilization as an amalgamation with

conventional or other herbal approaches, or as a

successful alternative for those who do not want to

risk undesirable side effects and can have improved

well-being despite illness.

Acknowledgement:

We appreciate all unknown referees for their

invaluable comments.

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Contribution of Individual Authors to the

Creation of a Scientific Article (Ghostwriting

Policy)

C. N. wrote the manuscript with input from all

authors. K. C. conceived and planned the

experiments. K.P. carried out the experiments. P.S.

and C.T. supervised the project.

Sources of Funding for Research Presented in a

Scientific Article or Scientific Article Itself

This work was funded by Eastern Herb. Co., Ltd

Conflict of Interest

The author has no conflicts of interest to declare.

Creative Commons Attribution License 4.0

(Attribution 4.0 International, CC BY 4.0)

This article is published under the terms of the

Creative Commons Attribution License 4.0

https://creativecommons.org/licenses/by/4.0/deed.en

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