The document summarizes the development of a Ganoderma lucidum extract herbal tea product with higher triterpene content. Key steps included: 1) Cloning the MVD gene from G. lucidum and constructing an overexpression vector containing the MVD gene driven by the gpd promoter. 2) Transforming Agrobacterium tumefaciens containing the overexpression vector and selecting strains with increased triterpene production. 3) Optimizing cultivation and extraction methods to produce a herbal tea product with concentrated triterpenes for potential health benefits. Efficacy and safety testing were also discussed to support marketing and commercialization of the product.

1. Chief Technical Officer：Jingyi Guan
Marketing Director：Xin Wang
Planting Happiness
1

2. TABLE OF CONTENTS
BACKGROUND INTRODUCTION
TEAM INFORMATION 4
PRODUCT SUMMARY 4
GANODERMA LUCIDUM 4
TRITERPENES 5
MEVALONATE PYROPHOSPHATE DECARBOXYLASE (MVD) 6
GROBACTERIUM TUMEFACIENS-MEDIATED TRANSFORMATION 8
GPD PROMOTER 9
SECTION A: RESEARCH, DEVELOPMENT AND PRODUCTION 6
CLONING FLOW CHART 11
HYPHAE COLLECTION 11
OBTAINING TARGET GENE MVD 11
CONSTRUCTION OF OVER-EXPRESSION VECTOR GL-GPE 12
OBTAINING AGROBACTERIUM STRAINS CONTAINING TI PLASMID GL-MVD 16
AGROBACTERIUM-MEDIATED TRANSFORMATION & SELECTION AND SCREENING 19
TRITERPENES OVER-EXPRESSION STRAINS 20
PASSAGE STABILITY 21
OPTIMAL TIME LENGTH OF PLANTING 21
PLANTING 22
SUPERCRITICAL CO2 FLUID EXTRACTION 23
ELECTUARY PRODUCT 23
SECTION B: EFFICACY AND SAFETY TESTING
EFFICACY TESTING 26
SAFETY TESTING 30
MAMMALIAN TOXICITY TESTING 31
ENVIRONMENTAL IMPACT 32
SECTION C: MARKETING
TARGET MARKET 34
MARKETING PLAN 34
YERHERB® IN THE MEDIA 35
COST ANALYSIS AND PRICING 36
CONSUMER PERCEPTION OF THE PRODUCT 37
REFERENCES 39
2

3. Background Introduction
3

4. Team Information
Mr. Ye, founder of Yerherb Inc., was born the rural areas of Guangdong, China.
At the age of 18, he started his career working at various traditional herb shops in
different large cities. Intelligent, hard working, honest, Mr. Ye was promoted to main
buyer position over the years. Due to the wars in Mainland China, Mr. Ye and his wife
fled to Hong Kong, subsequently raising a family of eight children there. By the time
Mr. Ye decided to immigrate to the United States, he had become a shareholder of the
herb chain he worked at.
When Mr. Ye moved to Los Angeles in 1979, he immediately saw the vast
business opportunity due to the lack of availability of traditional Chinese herb in
United State. Yerherb officially opened its first storefront as a small family operation.
Its reputation grew rapidly in the US, the herb products sells many places in US.
Jingyi Guan and Xin Wang was entered in this company in 2005, they were in a
project group developing the Ganederma lucidum herbal tea product that will be talk
in details below.
Product Summary
The product produced is a Ganoderma lucidum extract herbal tea, with multi-
functions on people health. It has higher concentration of active ingredient and the
cost of the product is lower than the Ganoderma lucidum products present in the
market now.
Ganoderma lucidum
Ganoderma lucidum or Lingzhi mushroom, which has a shape of semi-circle or
kidney-like, is a sort of medical mushroom and has been used as popular remedy in
traditional Chinese medicine for more than 2,000 years. In most of Chinese ancient
medical works, we can see the similar description about G.lucidum-------a super
panacea. It is effective for treatment or prevention of neurasthenia, high blood
pressure, arrhythmia and enhancement of resistance of various diseases. What is
4

5. more, from recent study, G.lucidum still has efficacy of curing cancer, diabetes and
even inhibiting senility. Nowadays the mushroom has been listed in the American
Herbal Pharmacopoeia and Therapeutic Compendium.
Triterpenes
Based on the previous study about the active ingredients in G.lucidum, the
efficacy mentioned to a great extent relates to chemical “triterpenes”. Triterpenes is a
series of secondary metabolites which belong to lanostane when the mushroom
forming fruiting body. At least 170 different triterpenes have been exacted from
G.lucidum so far. Here is a image of the structures of three typical triterpenes (or
ganoderic acid).
5

6. Triterpenes can inhibit secretion of histamine from mast cells which can cause
allergy and inflammation. What is more, ganoderol A, ganoderol B, ganoderic acid Y
which belongs to triterpenes can robustly inhibit cholesterol synthesis by interrupting
the synthetic pathway from acetate to cholesterol. Fe2+
—ascorbic acid which can
cause peroxide of lipid and 1,2,3-Benzenetriol which cause oxidation of erythrocyte
membrane can also be inhibited by triterpenes. In recent study, triterpenes has been
known that has effect in suppressing the growth and proliferation of cancer cells and
inducing apoptosis in a variety of leukemia, lymphoma, and myeloma cells by
increasing the amount of T lymphocyte and enhancing the activity of NK cells, IL-2,
tumor necrosis factors and macrophages.
However, the content of triterpenes is very low in wild-type G.lucidum, which
results in high cost in pharmaceutical industry.
Mevalonate Pyrophosphate Decarboxylase (MVD)
6

7. Triterpenes is synthesized through the MVA (mevalonate) pathway. (Figure) And
during the pathway, a lot of enzymes,including HMGR,FPS, SQS and MVD, show
essential functions. Among these enzymes, MVD (mevalonate pyrophosphate
decarboxylase) is a key enzyme in catalyzing mevalonate-5PP into isopentenyl-PP
within the process of synthesis of Triterpenes.
7

8. MVA pathway
1. Acetyl-CoA acetyltransferase, AACT; 2. 3-hydroxy-3-methylglutary-CoA synthase, HMGS;
3. 3-hydroxy-3-methylglutary-CoA reductase, HMGR; 4. mevalonate kinase, MK;
5.phosphomevalonate kinase, MPK; 6. pyrophosphomevalonate decarboxylase, MVD; 7.
isopenteny-diphophate isomerase, IDI; 8. famesyl diphosphate synthase, FPPs; 9. squalene
synthase, SQS; 10. 2, 3-oxidosqualene-lanosterol cyclase, OSC;
11. geranlgeranyl-PP synthase, FPS
Gene mvd which encoding protein MVD（Genbank: HQ5964）, and has a not
very long length of 1203bp. The complete sequence which is crucial for obtaining or
amplifying the gene has been known nowadays.(figure)
Agrobacterium-Mediated Transformation System for Fungi
Tumor inducing plasmid (Ti) is a kind of vector that originally used in
Agrobacterium-mediated transformation to plants. Target gene could be inserted into a
region called ”T-DNA” on the Ti plasmid which would be integrated into genome of
8
Sequence of gene mvd from Genbank

9. the Agrobacterium-infected plant cells by recombination. Now of Agrobacterium-
mediated transformation system for fungi has been well-established and shows a very
good transforming efficiency by modifying the T-DNA sequence to make it include
homologous region with fungi. Additionally, specific selection markers and promoters
to fungi are utilized in such a system.
Gpd Promoter
9
Agrobacterium-mediated transformation
A: Agrobacterium tumefaciens B: Agrobacterium genome C: Ti Plasmid : a: T-DNA , b: Vir
genes , c: Replication origin , d: Opines catabolism genes D: Plant cell E: Mitochondria F:
Chloroplast G: Nucleus

10. Strong promoters are the sequences that have high affinity with RNA polymesare
and can promote synthesis of mRNA highly efficiently. gpd (glyceraldehyde 3-
phosphate dehydrogenase,GAPDH) promoter is a strong promoter in large
filamentous fungi which can be used for constructing the over-expression vectors. The
sequence of the promoter has been known by cloning the 5’ flanking region of the gpd
gene.
10

11. Section A: Research, Development
and Production
11

12. Research and Development
Cloning flow chart：
Hyphae Collection
Before all the experiments we would conduct, tissues of the fungi in specific
stage which would produce mRNA we wanted should be obtained. The wild type
strain G20 will be cultured in the PDA liquid medium, which with 150rmp shaking
bed incubation for 7 days under 28 . Then the hyohae could be collected by gauze.℃
Obtaining Target Gene mvd
To obtain the target gene mvd, isolation of total RNA for synthesis of cDNA
should be accomplished first. Our team used liquid nitrogen to grind hyohae. After
homogenization, chloroform and isopropanol were used for isolation of RNA. The
isolated RNA was purified by Dnase and phenol. The quality of the total RNA was
proved good based on the result of gel electrophoresis.
The total RNA we acquired were then utilized in synthesis of cDNA by reverse-
12

13. transcription. For the reason that fungi is eukaryote which has poly(A) tail at 3’end of
mRNA, Oligo(dT) and reverse-transtripatase (M-MLVRTase) were used respectively
as primer and DNA polymerase during the reaction.
The cDNA products we obtained is a mixture of cDNA from different mRNA. To
get specific cloning product of mvd gene, we designed a pair of primer(named MVD-
O1 and MVD-O2) based on the cDNA sequence from GenBank (No.HQ596495.1).
Furthermore, we introduce two restriction sites respectively into these two primers
(sequences with underlines):
MVD-O1: 5’- ACTGggatccATGAGCGTATACCAAG-3’ (Bam HI)
MVD-O2: 5’-ACTGtctagaTCACTTCGGAAGGCC-3’ (Xba I)
Then we conducted PCR for specific amplification of mvd gene using the MVD-
O1 and MVD-O2 primers and total cDNA and successfully obtained target gene mvd.
Construction of Over-Expression Vector GL-GPE
Before construction of the over expression vector GL-GPE, strong promoter gpd
is needed to be purified from total DNA and specifically amplification. Based on the
sequence of gpd we got from other lab, we designed the specific primers GL-GPD-F1
and GL-GPD-R1 and introduce BstX1 site and Aat II site respectively into the
flanking region of the primers.The regions with underlines are BstX1 site and Aat II
site.
GL-GPD-F1:5’-GATCgacgtcTCCAAAGCCGCTCTCATGG-3’ (Aat II)
GL-GPD-R1:5’-GATCccaacatggtggAGGGGGATGAAGAGTGAG-3’ (BstX I)
After we obtained the specific amplified products, we created recombinant
plasmid PGL-GPD with plasmid pCAMBIA 1300.
13

14. pCAMBIA 1300 plasmid is a Agrobacterium binary vector for plant or fungi
transformation. This plasmid is a shuttle vector which can be replicated and expressed
14
Diagram of pCAMBIA 1300 plasmid

15. in both E.coli and Agrobacterium. It has two selective markers, one is hgyR
(hygromycin resistance) which is used for plant or fungi cells selection and the other
marker is KanR
(kanamycin resistance) which is used for bacteria cells selection. We
digested the pCAMBIA 1300 plasmids and gdp promoter fragments with BstX1 and
Aat II. Then we used T4 ligase to ligate the pCAMBIA 1300 plasmid vector and the
gpd fragments. And we got ligation product pGL-GPD.
Competent E.coli Cells were created by heat shock with Ca2+
and mixed with
15
Construction of binary vector pGL-GPD
0 Aat II

16. ligation product we got from last step. Then we incubated the solution and plated the
solution onto kanamycin LB medium for selection of transformant colonies. After
incubation, we took individual colonies on the plates, purified plasmid from cells
using kit and did RFLP (restriction fragment length polymorphism) for screening.
Within the screening, we extracted the plasimds from transformant cells. After
digestion by BstX I and Aat II, we did agrose gel electrophoresis with EtBr and
identified the recombinant colonies which can show a band of inserted fragment. Then
we cultured cells from those colonies and isolated vectors pGL-GPD. Here is the gel
electrophoresis result.
The inserted gpd
promoter replaced the
CAmv35S promoter
which was used for
expression of higher
plant genes. But the
plasmid still needed another gpd promoter with different direction for following
inserted target gene expression. For that, we designed two primers for specifically
amplification gpd promoter fragments with two different restriction sites (which were
located in MCS):
GL-GPD-F2: 5’-GATCggtaccTCCAAAGCCGCTCTCATGG-3’ (KpnI)
GL-GPD-R2: 5’-GATCgaattcAGGGGGATGAAGAGTGAG-3’ (EcoR I)
Same method of digestion and ligation of inserted fragments and plasmids were
used here as above with enzymes KpnI and EcoR I.
After transformation and selection for transfromants, we did PCR screening and
agrose gel electrophoresis using GL-GPD-F2 and GL-GPD-R2 primers for obtaining
16
The digestion of plasmid pGL-GPD by BstX I and Aat II
1,2: pgl-GPD; 3,4: The digestion by BstX I and Aat II; M: DL2000 DNA Marker

17. the recombinant starins. Then we cultured the strains and isolated over-expression
vector named GL-GPE.
17
Amplification pattern obtained with primers
for fragment gpd from transformant
1-3: randomly chosen transfromants; P: GL-GPE as postive control; N:
untransfromed E coli as negative control; M: DL 2000 DNA Marker

18. Obtaining Agrobacterium Strains Containing Ti Plasmid GL-MVD
In construction of mvd over-expression plsamid GL-MVD, GL-GPE vector and
mvd cDNA fragment was digested with Bam HI and Xba I and ligated with each other
by T4 ligase.
18
Construction of over-expression vector GL-GPE

19. After using heat-shock with CaCl2 to make competent agrobacterium, GL-GPE
solution were mixed with the cells. Because the Plasmid has a kanamycin resistance
selection marker, we used LB medium with kanamycin to select transformants.
Then we
took
transformant
colonies and
19
Bam HI and Xba I digest
Full-length mvd gene
Construction of over-expression vector GL-MVD
Selection for transformants on kanamycin medium

20. isolated plasmid which was used for RFLP (restriction fragment length
polymorphism) screening. During the process of RFLP, we digested the plasmids with
BamH I and Xba I and conducted the agrose gel electrophoresis. The recombinant
colonies were identified when the band of inserted mvd gene (1203bp) shown on the
gel.
Agrobacterium Tumefaciens-Mediated Transformation
Before transformation of the GL-MVD plasmid into G20 Ganoderma lucidum
strain, protoplasts of the fungi were prepared. The grinded hyohae was treated with
lywallzyme to remove cell wall. Then the solution of Agrobacterium was mixed with
the protoplasts and incubated for infection. The mixture then was plated on
hygromycin PDA medium to select transformants.
20
The digestion of plasmid GL-MVD byBamH I and Xba I
1, 2: double restriction enzyme digestion of plasmid GL-MVD; M:
DL 2000 DNA Marker

21. And then these tranformants were subjected to chromosome DNA extraction for
PCR screening with gpd promoter primers GL-GPD-R2 and mvd primer MVD-O2 to
test if the fragment has been inserted into chromosome genome and if the gpd
promoter has fused with gene mvd. After obtaining amplification products, we did
agrose gel electrophoresis to identify recombinants which shown the band of inserted
mvd gene. And we named those recombinant strains “GMOEi”(i=1, 2. 3.......15)
(Figure)
21
Amplification pattern obtained with primers for inserted gene mvd
in genomic DNA isolated from GMOEs.
Lane 1, negative control with no template DNA; Lane 2, negative control with
untransformed G.Lucidum; Lane 3, GL-MVD as positive control; Lane 4 to 18,
GMOE1 to GMOE15; Lane M: DL 2000 DNA Marker
Selection of hygromycin-resitant transformants of G.Lucidum
1,5: G20 as the negative control; 2, 3, 4, 6, 7, 8: The selected hygromycin-resistant
transformants

22. Triterpenes Over-Expression Strains
After culturing such recombinant strain for 20 days, we tested and compared the
triterpenes contents of the over-expression strains with wile-type. In this experiment,
we used ultrasonic method to bread the cells and extract triterpenes by chloroform.
Then we used the vanillin perchloric acid colorimetric method to measure oleanic acid
content for establishing standard curve in standard solution and triterpenes content in
the strains by testing OD values under A245nm. At last, we found that triterpenes
content of one of the over-expression strains GMOE10 which was 3.5mg/100mg dry
weight was 101% more than the content of wild-type (G20).
Passage Stability
To ensure the passage stability of the insertion, we did subculture of the
GMOE10 strain respectively for 3/5/10 generations and test the triterpenes content.
The results of subculture show that triterpenes over-expression phenotype of the
GMOE10 was stable from generation to generation.
22
Detection of the triterpenes of GMOEs
Content of triterpenes at different generation

23. Number of generations Content of triterpenes(mg/mg dry weight)
3 3.6
5 3.4
10 3.5
Optimal Time Length of Planting
Though we obtained the over-expression strain, it was still not enough for
optimize triterpenes producing for the reason that the secretion of triterpenes are
different at different stages of life cycle of Ganoderma lucidum. So we designed an
experiment to find out the optimal time length of planting. Here we adopted the well-
developed planting method as in agricultural industry to culture the GMOE10 strain.
sawdust, bran, sucrose, gypsum, water will be mixed following a ratio equal to
73:25:1:1:65 and filled into LDPE bag as compost. The compost were then sterile and
adjusted to pH 8.0. After planting the 20-day GMOE10 mycelium in different bags,
we marked those bags with “15 days”, “35 days”, “55 days”, “75 days”, “90 days”
which meant different time length of growing for different bags. These mycelium
contained bags were incubated under 27 and 90% air humidity. (image)After℃
finishing incubation, we extracted triterpenes using ultrasonic and chloroform and
measured the contents of triterpenes of different samples. The result indicated that
optimal time length of planting was 75 days.
Number of days for planting Content of triterpenes(mg/mg dry weight)
15 2.8
35 4.1
55 6.5
75 9.7
90 8.1
Scale-up Production
23
Contents of triterpenes of incubation of different time length

24. Planting
To obtain enough Ganoderma lucidum as raw material, we built up green houses
for large-scale planting. Same method including 75-days culturing as what we
mentioned above was used for planting. Additionally, beside temperature and air
humidity, light was another factor needed attention. Too strong or too weak light
would inhibit the growth of the fruiting body of the fungi.
Supercritical CO2 Fluid Extraction
To further enhance the efficiency and safety and lower the cost and
environmental contamination of triterpenes extration in Industrial production, we
24
Planting G.lucidum

25. decided to adopt the supercritical CO2 fluid extraction （ SFE ） method. Such a
method is based on the principle that when the supercritical CO2 fluid gets in touch
with and dislsoves the mixture, compositions which have different polarities, boiling
points and molecular weights could be separated under different pressures and
temperatures. Then when the fluid returns to CO2 gas, the extract could be
precipitated. Because the method works under a temperature range from 35 to℃
40 , the volatile compositions could effectively maintain after extraction. We℃
purchased a 40L SFE equipment which combined with ultrasonic breaking from
Taiwan Supercritical Technology CO., LTD, and such mixed extraction method
provided a dramatically enhancement in efficiency of extraction from 3.5% to 41%.
Electuary Product
To produce electuary for G.Lucidum tea, the extract was evenly mixed with
dextrin with a ratio of 1:20 to make extractum. After squeezing trough the screen
mesh (mesh number=20) to make powders, 70 heat was used to dry the powders℃
and finished products were packaged. And we also produced canned drinks from dry
25
40L SFE equipment
40L SFE equipment40L SFE equipment

26. powdered.
26
Image of our products

27. Section B: Efficacy and Safety
Testing
27

28. Efficacy Testing
Ganoderma lucidum extract on mice test approved by FDA
The effects of Ganoderma lucidum extracts on the immune function and growth of mice
under the experimental incubation were studied. The results showed that the weight of mice
gained in the testing groups were higher than the control groups after they fed on the extracts,
and their abdomen phagocytic index and phagocytosis rate of macrophages increased
significantly after they fed on the extracts for 30 days and did injections for 7 days. The rates
between the mice 's liver and spleen weights to their body weights increased compared with
the negative control. The results suggested that Ganoderma lucidum extracts enhanced the
mice 's immune function.
The material and protocol for the test approved by FDA:
Preparation of the chicken blood with red blood cells
Extract 1ml chicken blood from the live healthy chicken wing. Transfer the chicken blood
into 5ml Alsvers solution. Made a 5% of the cells suspension with sterilized saline.
Preparation of Ganoderma lucidum extract water used in our product
Method used was same as our product extraction as previously discussed.
Testing on different groups of mice
The animals used in this experiment are pure white mice, weight 16g-18g. These mice
were divided into 6 groups at random, each group of 10, with free feeding standard feed.
Group 1 ~ 4 group drank Ganoderma lucidum extract water, the concentration respectively 3.
2 mg/mL, 11. 2 mg/mL, 32. 0 mg/mL and 140 mg/mL, in feeding after 30 days, testing
28

29. groups of mice did celiac injection with the different concentrations of Ganoderma lucidum
extract of 1 mL/day for 7 days. Group 5 was negative control group, which drank distilled
water. 30 days after, they were injected of saline by 1 mL/day for 7 days. The group 6 as the
positive control group, which had the same breeding method as the negative control group.
Before two days of the death of the mice, the abdominal cavity injection was performed on
them with cyclophosphamide (final concentration of 0. 75 mg/m, L), 1 mL/day, continuous (2
days). In 24 hours after the last injection, they were executed by cervical dislocation method.
Recorded each feed quantity and water quantity, and weighted each mouse in different
groups, calculated the mean of each group.
Mice abdominal cavity macrophage specimen preparation
Mice treated by intraperitoneal injection of 5% chicken blood red blood cells, each mouse
injected by 0.5ml. The cervical dislocation method executed after 2 hours of injection.
Instantly by intraperitoneal injection of 0. 5 mL saline, gently rubbed the abdominal cavity
for 1 min, cut open abdominal skin, and made a small cut in muscle layer, transferred
peritoneal fluid on the clean slide, incubated for 30 min at 37 . Using 10% Giemsa staining℃
before observe under the microscope. Direct observed under microscope, there were 2000
macrophage in each mouse. The phagocytosis rate and phagocytic index were being
calculated:
Phagocytosis Percent = (phagocytose chicken blood red blood cell number of macrophages
present 2000) x 100%
Phagocytic index = total number of chicken blood red blood cells being phagocytized ÷
number of macrophages present phagocytose chicken blood red blood cells
Determine the ratio of the organ weight to weight of the mice
After executed of the mice immediately, opened the abdomen and chest, quickly removed
the liver, lung, spleen, thymus, they were weighing respectively. Calculated organ to body
weight ratio and the average value of 10 mice in each group.
29

30. Result and Discussion:
Effect on mice abdominal cavity macrophage phagocytosis
Microscopy results show in table1, four different concentrations of Ganoderma lucidum in
experimental mice abdominal cavity macrophage phagocytosis rates compared with negative
control group have obvious improvement, increased in 20. 16% ~ 69. 79%, and got a significant or
extremely significant difference. The concentration with 3. 20 mg/mL Ganoderma lucidum group
phagocytosis rate increased the most, the rest of the groups phagocytosis rate increased slightly.
Positive control group was lower than negative group by 42. 99%. This shows that Ganoderma
lucidum extract water had enhanced the experimental mice abdominal cavity macrophage for
chicken blood red blood cells phagocytosis rate, improved the cellular immune function of mice.
Table1: Effect on mice abdominal cavity macrophage phagocytosis
Test group Concentration
(mg/ml)
Phagocytosis
rate(%)
Phagocytosis
index
Negative group 0 25. 53± 0. 67 1. 50± 0. 21
Positive group 0 14. 55± 1. 84 1. 28± 0.13
Ganoderma lucidum groups 3.2 43. 34± 2. 11 1. 34± 0. 20
Ganoderma lucidum groups 11.2 38. 37± 1. 49 1. 42± 0. 04
Ganoderma lucidum groups 32.0 35. 69± 0. 89 1. 67± 0. 03
Ganoderma lucidum groups 140.0 30. 67± 0. 714 1. 54± 0. 11
From the data shows in table 2, by feeding with Ganoderma lucidum extract water, the mice
growing average weight is greater than the control group, different concentration of Ganoderma
lucidum group had different weight gain. The concentration with 3.2 mg/ml increased the most.
The highest concentration of treatment group, weight gain was not the most. The reason might be
that with the increase of Ganoderma lucidum component content in extract solution create the
bitter taste of the drinking water, make high concentration group water consumption lower than
the control group and low concentration group, which affect weight gain.
Table 2 shows the effect on the body weight of the mice
Test group Concentration
(mg/ml)
Average of Body
weight in the group
(10 mice)
Percentage of
increase (%)
30

31. Negative group 0 0. 1479 ---
Ganoderma lucidum groups 3.2 0. 2382 61. 05
Ganoderma lucidum groups 11.2 0. 1684 13. 86
Ganoderma lucidum groups 32.0 0. 1719 16. 23
Ganoderma lucidum groups 140.0 0. 1924 31. 30
As shows in table 3, mice in different concentrations of Ganoderma lucidum groups by drinking
Ganoderma lucidum extract water after 30 days and 7 days of injection, the liver, spleen and
thymus to body weight ratios were significantly increased than the control group, and had no
obvious adverse effect on the lungs. Spleen and thymus are immune organs, the liver has
detoxification function. From this result, the long-term use of a certain amount of Ganoderma
lucidum extract solution can improve the body's detoxification ability. In this experiment, the
development of immune organs in mice had promoted effectively, it had the effect that increased
in the body's immune function.
Table3: Effect on the ratios of organs to the body weights.
Test group Concentration
(mg/ml)
Liver/body
weight
Spleen to
body/ weight
Lungs/body
weight
Thymus/body
weight
Negative group 0 4. 59± 0. 49 0. 60± 0. 02 0. 67± 0. 06 0. 24± 0. 02
Positive group 0 5. 54± 0. 10 0. 38± 0. 01 0. 60± 0. 07 0. 16± 0. 05
Ganoderma lucidum groups 3.2 5. 14± 0. 55 0. 72± 0. 02 0. 69± 0. 04 0. 19± 0. 04
Ganoderma lucidum groups 11.2 5. 29± 0. 48 0. 89± 0. 08 0. 73± 0. 21 0. 23± 0. 12
Ganoderma lucidum groups 32.0 5. 76± 0. 38 0. 80± 0. 12 0. 66± 0. 05 0. 33± 0. 12
Ganoderma lucidum groups 140.0 6. 24± 0. 80 0. 87± 0. 10 0. 70± 0. 01 0. 19± 0. 13
Safety Testing
There are two types of safety tests were performed. They are ELISA and PCR tests. The FDA
required manufactures to to label their products with regards to eight specific allergens: milk,
31

32. eggs, fish, shellfish, peanuts, wheat, soybeans and tree nuts since 2005. These allergens are
responsible for over 90% of the documented food allergen-related cases. The most common and
preferred methods approved by FDA for allergens testing in food industry are ELISA and PCR.
The testing methods have been developed that can now detect allergens in finished products
even at very low concentration as per million (ppm) range. By performing this test, our product
can be detected if it contains any popular allergen at the molecular level. The allergens then can be
avoiding during manufacturing or being labelled on the product box for alert.
ELISA methods detect the actual allergen protein molecule by binding antibodies to the allergen
and then using an enzyme-linked conjugate to create a colorimetric change that can be measured.
The PCR methods, which are more sensitive and detect the DNA molecules of these allergens,
can be used in raw and finished products and are not affected by the heating process, because
DNA typically remains intact after being exposed to the high temperatures most foods. As food
allergens are becoming an increasingly important issue in food safety. These two tests are crucial
before the food products hit the market. Form the results of ELISA and PCR test. There was not
any popular allergen ingredient being detected in our product.
Allergen tests
Popular allergens ELISA PCR
milk None None
eggs None None
fish None None
shellfish None None
peanuts None None
soybeans None None
tree nuts None None
Ganoderma lucidum has been used in China for long time, there are several beneficial effects of
Ganoderma lucidum have been claimed. The majority of these claims have not been studied in
controlled clinical trials, but there has been an abundance of clinical use, as well as in vitro and
animal testing data support its safety. The safety of using it is further supported by common use of
Ganoderma lucidum as an edible mushroom and broad exposure to consumers with no adverse
effects reported.
32

33. Mammalian Toxicity Testing
In 2012, a double-blind, placebo-controlled, parallel group interventional trial was performed
by using our product. There were 50 generally healthy volunteers (age 18-65 years, BMI 19-35)
were administered beverages containing placebo (control) and our herbal tea includes Ganoderma
lucidum active ingredient (1.5g/day) for 12 weeks. After drinking for 12 weeks, there was no
adverse effect on both groups. The group consumed the active ingredient reported they got better
sleep, and felt the herbal tea reduce their feeling of stress. The BMIs for test group remained and
for some obese people their BMI reduced. Some people reported their skin looks healthier. The
results of this study indicate that Ganoderma lucidum at a dose of 15 g/person/day was functioned
in adults in the general population without any adverse effect.
Functional trial on 50 volunteers
Environmental Impact
Our product is pure Ganoderma lucidum extract powder with dextrin. Both ingredients were
approved by FDA to use in the food industry. From the experiment test on animals and allergen
tests by ELISA and PCR, there was no undesirable side effect for healthy adults. And as our
planting environment is in the greenhouse under very restrict controls, there is not any negative
effect to the environment. The species is also planted in America continent for a long time in some
area. So there is not any issue with biological invasion.
Adverse
effects
Sleep better Feel less
stressful
Better looks
skin
Getting
infected by
disease during
12 weeks
Blood
cholesterol
levels
reduced
Control
group
None 3 people
reported
2 people
reported
4 people
reported
8 people
reported
2 people
reported
Testing
group
None 42 people
reported
38 people
reported
39 people
reported
2 people
reported
35 people
reported
33

34. Section C: Marketing
34

35. Target Market
Our product is facing healthy or sub-healthy state adults and seniors. The product is designed
for people living in stressful environment. These people may associate with sleeping disorder,
anxiety and immune function reduce. The product is also aim to people with chronic alcohol
drinking habit. These people may have higher blood pressure and unhealthy liver, which can be
reduced by keep drinking our product. Our product also designed to all kinds of people want to
keep a good look skin, as it has anti-aging function by keeping consuming it. It is also applicable
to vegetarians.
Marketing Plan
Step1: set up the goal as achieve sales 50,000 boxes of product per month within six months.
Step2: evaluation of the internal situation of the business marketing the product
• Competition: our competitors could be supplements products, medicines, and other
functional herbal drinks.
• Our advantages: Our product is multi-functional herbal tea, which not includes any
harmful product. It is very easy to take and it taste better. During the production it has the
lower cost compared to the product exist in the market in nowadays. Our product has
much higher concentration of the active ingredient, and our pricing is better for the
consumers.
Step3: Description of the ideal customer for the product including age, household income,
geographic location, work situation.
Our ideal customer are healthy and sub-healthy adults or senior people with higher incomes. The
product is aim in large cities in US.
Step4: Create marketing strategies.
There will be several types of promotions discussed as following.
Step5: Create the marketing budget.
The cost will be talked in details later.
35

36. Yerherb® in the Media
There will be several promotions of this product to be well known and reach
our target sales goal. At the beginning of the sales, the TV advertisement and
Radio advertisement of the brand and the product will be distributed for familiar
and well known. The mainly information of the advertisement is about or brand
name and the multi-functions of this new kind of herbal drink.
As our pricing is higher than regular herbal teas. Our herbal tea product with
several functions will aim to people have higher consumption ability. For this
reason, there will be brochures distributed in the airport and on the flights to
reach more high income costumers. There will also be online video
advertisements, as people using computers may have issues with sub-healthy are
our major target costumers.
The posters will be shown in poplar pharmacies and markets. And there will be
some free samples provided to the consumers interested in the product. There will
be some sales promoters in the market to introduce the background and function
of our product to consumers in details.
36

37. Brochures
37

38. 38

39. Poster
Cost Analysis and Pricing
The equipment will produce 500 tons Ganoderma lucidum extract from 2,200 tons of raw
materials in each year. There will be 40 greenhouses, 7 acres in total for the planting of the
Ganoderma lucidum raw material. For each greenhouse, it will produce about 600
Ganoderma lucidum plants. The total price of the planting is about $500,000 per year. The
science and development cost will be $150,000 per year (Chemical reagent, Enzymes and
vector, PCR & post-PCR analysis, G20 strain, lab equipment). The dextrin used costs about
$200,000 per year. The cost of the processing equipment will be $100,000 for once. The
maintenance and labor cost for each year is about $100,000. The logistics of our product is
about $500,000 per year. The promotion and advertising costs are $1,000,000 dollars per
year. Our product is 28 tea bags per box (approximately 30g/ bag). The price of our product
in the marketplace will be labelled as 16 dollars per box. We will also have canned
drinks，which includes 30g of active ingredient plus water per can in 350ml. This is better
for the people who do not like hot drinks. The canned drinks are very convenient to carry and
drink at any time for the consumers. The price for the can drink will be 4 dollars per can.
Image of tea bag
39

40. image of canned drink
Consumer Perception of the Product
The existing herbal teas in the market are approximately 6 to 7 dollars per box.
They could be our major competitors. As most of the herbal tea in the market now
40

41. only has one or few functions or just with some herbal or fruit flavors, they
cannot be considered as functional herbal tea. Our product includes very precious
ingredient. Which has the same value as ginseng, cubilose. By doing market
research, most of ginseng tea in market in US are imported. It is difficult to get
them in America’s popular supermarkets. Our product is planted in the US and
compared to other precious herbal product it has higher active ingredient. It is the
best choice for the people to consume with long time to maintain their health and
enjoy the herbal flavor drinks.
41

42. Reference：
1.Shi,L., Qin,L., Xu,Y., Ren,A., Fang,X., Mu,D., Tan,Q. and Zhao,M. (2012).
Molecular cloning, characterization, and function analysis of a mevalonate
pyrophosphate decarboxylase gene from Ganoderma lucidum. Mol. Biol. Rep. 39 (5),
6149-6159
2.Shi,L. (2012). The developmet of agrobacterium tumefaciens-mediated
transformation and its application in the study of triterpenes biosynthesis of the
meidical fungus Ganoderma lucidum [D]. Nanjing Agricultural University.
3.Cheng, C., Yue, Q., Wu, X., Wu, Z., Song, X., Tao, S.. . Guo, D. (2010). Cytotoxic
triterpenoids from Ganoderma lucidum.Phytochemistry, 71(13), 1579-1585.
4.Sliva, D. (2006). Ganoderma lucidum in cancer research.Leukemia Research, 30(7),
767-768.
5.LI Xiufen, QIAN G Yufeng , YI Huilan, LI Xianlei ( School of Lif e Science and
Technology , Shanx i University , Taiyuan 030006,China
42