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Abstract: We set out to discover if chemotypes exist in Artemisia species, how artemisinin concentration changes with habitat 

height, and whether there are any new plant sources of artemisinin that may replace Artemisia annua. Research Tools and 

Procedures: Seven species, spanning three distinct height ranges, were chosen for this investigation. We used high-performance 

liquid chromatography to quantify artemisinin after extracting it from the leaves, stems, and roots of every species of Artemisia. 

End result: The artemisinin content of various Artemisia species was shown to be significantly affected by their height variation. 

The artemisinin content of leaves increased at height II in three species of Artemisia: Artemisia moorcroftiana, Artemisia vestita, 

and Artemisia roxburghiana var. roxburghiana, with values of 0.09%, 0.08%, and 0.07% of dry weight, respectively. Artemisia 

vulgaris had a high artemisinin concentration at heights II and III (0.06% and 0.07% of dry weight, respectively), in contrast to 

Artemisia sieversiana, which exhibited a low artemisinin content at height I (0.08% of dry weight). Findings: This research found 

that chemotypes exist in Artemisia species and that plant geographical habitat height impacts artemisinin production.  

 

Keywords: Artemisinin, chemotype, ecotype, and height variation are some of the keywords regarding alternative species. 
 

 

Chinese Traditional Medical Journal 

 
The Role of Habitat Elevation in Artemisia Species Chemotype and Artemisinin 

Production 

Rajkumar 

Department of Pharmacy, COMSATS University Islamabad, Abbottabad Campus, Abbottabad, Pakistan 

Received on:  21 Dec 2024   Revised on: 20 Jan 2024    Accepted Date: 25 Feb 2024  
Published on: 04 April 2025 

 

 
 

 

INTRODUCTION 
 

The Asteraceae family annual plant Artemisia annua, which 

has a long history of use in Chinese medicine for the 

treatment of malaria, produces artemisinin, a potent weapon 

against parasites that have developed resistance to traditional 

treatments. [1] Optimal effectiveness in combating this illness 

and lowering transmission rates is now achieved by 

combining artemisinin with other medications. [2] in 

Artemisia is most abundant in cold and temperate zones in 

North America and Eurasia, especially in the temperate belt 

of Asia. The following nations are significant: Spain, 

Argentina, France, America, Bulgaria, France, and Hungary. 

the third Notably, it is suggested that northwest Asian 

mountain territories (such as mesothermic subarctic or 

semi-humid forest steps near Urals) encompassed genus 

origins based on modern distribution patterns relevant to 

fossil data;[4] primary types being situated mostly in the north 

 

The amount of artemisinin in different Artemisia species is 

reportedly unpredictable and may change over time, even within 

the same plant. The natural population of A. annua, on the other 

hand, contains very little artemisinin (0.01%-0.15%). [5] The 

artemisinin content of plants in this species may vary due to 

factors such as cross-pollinating, with larger concentrations seen 

during full bloom. Other factors that greatly impact the 

production levels of mature plants include seasonal variations and 

geographical location. Thus, variations in the amount of 

artemisinin found in A. annua leaves are probably attributable to 

variances in harvest time or different cultivars employed, as well 

as environmental stress factors like light exposure or nutrient 

availability, which might affect the productivity of these plants. 

 
 

temperate. This suggestion is mainly based on the modern  

distribution of Artemisia and its allies, with their primary types 

mostly lying in north temperate Asia, as well as on fossil and 

paleogeographic data. 

 

 

 



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There has been debate in the literature on which plant parts 

have the greatest concentration of artemisinin. There is ten 

times more artemisinin in flowers than in leaves, say the 

researchers. [6] Nonetheless, there are some who argue that 

the leaves contain about 89% of the total, with the tallest 

region reaching a maximum of 41.7% at 50 cm, almost 

double the amount found in the lower portions. There is a 

wide range of out-of-leaf collection rates among frequently 

cultivated accessions, from 0.02% to 1.38%, as reported in 

the literature. These results point to diverse results, thus 

researchers are looking at measuring levels in different parts 

of plants at different altitudes (roots, stems, and leaves). 

Distinct total content distributions depending on leaf 

components were found in lowland and highland locations 

when compared. This sparked more studies with the goal of 

improving yields worldwide. Despite their heavy reliance, 

institutions in Pakistan's northern hilly areas have mostly 

ignored biodiversity reserves, highlighting an opportunity to 

promote ecological and economic advantages. Everyone 

agrees that it's critical to stress sustainability via appropriate 

activities that don't upset the ecological equilibrium. The 

significance of teaching children about different viewpoints 

on human relationships, including living in harmony with the 

natural world, is highlighted by this method. As a result, it 

has risen to the forefront of educational discourse, calling for 

multidisciplinary groups to investigate and use the best 

scientific methods. Creative solutions may be fostered via the 

integration of conventional knowledge with culturally aware 

approaches through these tactics. The artemisinin 

concentration of Artemisia annua, a plant known for its 

powerful antimalarial qualities, has been shown to vary 

according to its growing height. Several studies have shown 

that artemisinin levels are directly correlated with elevation; 

plants grown in certain altitude ranges tend to have much 

higher concentrations of this bioactive component. Due to 

variations in environmental conditions such soil composition, 

sunshine exposure, and temperature, it is generally believed 

that A. annua specimens with higher heights have higher 

levels of this important chemical. When it comes to drug 

manufacturing, increasing yields is of the utmost importance, 

and altitude gradients play a significant role in this endeavor 

by influencing physiological processes associated with 

secondary metabolite production, such as the pathways that 

lead to substances like artemisinin. 

 

MATERIALS AND METHODS 
Plants collection 
Seven Artemisia species that occur naturally in Northern 

Pakistan were gathered from various hilly locations [Figures 

1 and 2]. All seven species were collected at three different 

elevations, where their population was observed to be highly 

dense. The elevations of the sampling points (in feet) and 

the height groups (categorized as I, II and III) of the seven 

Artemisia species are presented in Table 1. A taxonomist 

identified these specimens using the Flora of Pakistan reference 

book at Quaid-i-Azam University’s Department of Plant 

Sciences located in Islamabad, Pakistan, by comparing them 

with already designated herbarium sheets for each individual 

Artemisia spieces kept within a preserved collection of dried 

plants housed there. We selected these particular seven wild- 

grown Artemisia varieties so we could measure artemisinin 

concentration levels present throughout leaves’, stems’ or 

root systems harvested from three elevation-based strata 

as measured against height I (<5000 ft), height II (between 

5000–6000 ft) and finally climatic zone given name Height 

III; which occurs above an altitude greater than six thousand 

feet above sea level. 

Preparation of extracts 
The herbs were utilized to extract artemisinin using the 

method as reported earlier with modifications.[7] First, their 

weights were measured, and then, they were ground with a 

mortar and pestle until a homogenous mixture was produced 

in 5 ml of high-performance liquid chromatography (HPLC) 

grade toluene. The samples underwent sonication for half an 

hour over ice, preventing any possible evaporation due to 

overheating before being centrifuged (2000 g at −8°C) for 

20 min so that artemisinin-rich toluene could be separated 

from cellular debris. Next, the supernatant was extracted 

and preserved within dram vials while pellets containing 

cell debris were resuspended in fresh 5 ml amounts, as seen 

fit by each researcher, and sonicated again before centrifuge 

treatments through which heavy particles settled into these 

pallets. The supernatant was carefully removed, and the cellular 

debris was resuspended in toluene. The mixture was vortexed 

and centrifuged again to extract more artemisinin from the 

material. The retrieved supernatants were amalgamated to yield 

pooled extracts for subsequent HPLC analysis. Subsequently, 

the extracts were air-dried to ensure retention during storage, 

facilitated by gradual temperature reduction to minimize the 

risk of fragmentation, and then stored under optimal freezing 

conditions (-20°C). 

The yield was estimated using the following formula: 

% Yield = 
Weight of dried extract ×100 

Weight of plant sample 

 

Quantification of artemisinin 
The quantification of artemisinin in the dried n-Hexane 

extracts was performed using HPLC following the protocol 

advised earlier with modifications.[7] An Agilent Technologies 

Zorbax SB C18 column (150 mm × 4.6 mm × 5 m) was 

utilized as the stationary phase with a mobile phase flow rate 

of 1 mL/min. The Diode Array Detector (G1315B-DAD) 

indicated maximum absorbance at 260 nm and the retention/ 

elution time peak for artemisinin occurred after approximately 

12 min. Artemisinin identification employed an authentic 



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Figure 1: Geographical distribution of plant species collected from different heights (graphical abstract). A. vulgaris: Artemisia vulgaris, A. moorcroftiana: 

Artemisia moorcroftiana, A. vestita: Artemisia vestita, A. sieversiana: Artemisia sieversiana 

 

Figure 2: Geographical map of Northern Pakistan showing plant collection points 
 

standard of the compound while injection volumes for 

both samples and standards were fixed at 20 L each. Six 

dilutions of standard solution from concentrations ranging 

between 0 and 500 g/mL were then injected one by one 

into HPLC before obtaining a calibration curve by plotting 

chromatographic peak area (mAU) against concentration. 

Data analysis showed that this linear response depicted in 

Figure 3 had an excellent correlation coefficient R = 0.9994 

due to the successful application of linear regression analysis 

on the equation obtained from the consulting calibration curve. 

Statistical analysis 
All the experimental data were analyzed statistically 

using analysis of variance and Fisher’s least significant 

difference (LSD) test. 

 

RESULTS 
The altitude of the plant habitat had a significant effect ( level 

0.05) on the artemisinin content of leaves and stems of the 

same Artemisia species growing at three different spots. The 



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Figure 3: Calibration curve of standard artemisinin 

 

effect of altitude was more prominent in the leaves and stem 

artemisinin content in five Artemisia species, i.e., Artemisia 

vulgaris, Artemisia moorcroftiana, Artemisia roxburghiana 

var. roxburghiana, and Artemisia vestita. All of the 

Artemisia species we looked at have rather modest 

artemisinin concentrations in their roots. Sampling at height 

II revealed the highest concentration of artemisinin in the 

roots. A. vestita, A. moorcroftiana, and A. roxburghiana var. 

roxburghiana had very high artemisinin content in its leaves 

when measured at height II. Similarly, Artemisia vulgaris at 

heights II and III and Artemisia sieversiana at height I both 

have high artemisinin leaf content. In addition, the 

artemisinin concentration of the leaves was twice as high as 

that of the same species obtained from lower elevations 

[Figure 4]. At height II, the leaves of A. moorcroftiana had 

the highest artemisinin content (0.09% ±0.01%), whereas 

the leaves of A. sieversiana and A. vestita, collected at 

heights I and II, respectively, had the second-highest 

artemisinin concentration (0.08% ± 0.01%). Leaves of A. 

vulgaris taken at height III had the highest concentration of 

artemisinin at 0.07% ±0.01%, as shown in Figure 4.  

The complete plant sample of A. sieversiana grown at height 

I had the highest average amount of artemisinin. At height 

II, A. vestita and A. moorcroftiana showed the second-

highest average artemisinin concentration. A. vulgaris 

samples collected at height II had the third greatest 

concentration of artemisinin, whereas A. roxburghiana var. 

roxburghiana samples obtained at height III had the same 

result [Figure 5].  

Figure 6 displays the findings of the calculation of the 

average artemisinin content in the leaves, stems, and roots. 

According to our research, the concentration of artemisinin 

in the leaves was higher than in the stems and roots at all 

plant habitat heights, reaching a peak at height II (Figure 6). 

At all three of their habitat heights, we found that the 

artemisinin distribution patterns in the stems and roots were 

quite comparable to those in the leaves. 

 

DISCUSSION 
Artemisinin has been an effective remedy against different 

pathogenic fungi of plants such as Rhizoctonia cerealis, 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 4: The content of artemisinin in the leaves, stems, and roots of the 
seven Artemisia species collected from three different altitudes (alphabetic 
on the bars are ranking orders after analysis of variance and least 
significant difference). A. moorcroftiana: Artemisia moorcroftiana, 
A. vestita: Artemisia vestita, A. vulgaris: Artemisia vulgaris, A. indica: 

Artemisia indica, A. sieversiana: Artemisia sieversiana, A. roxburghiana: 

Artemisia roxburghiana 

 

Gaeumannomyces graminis var. tritici, Verticillium dahlia, 

and Gerlachia nivalis, suggesting a potential protective role of 

artemisinin against plant diseases.[8] Amusingly, in an era when 

the scientific community is striving hard to search for novel 

compounds with more specificity to their cellular and molecular 

targets, artemisinin stands prominent with its diverse molecular 

targets. Since the time artemisinin was recognized for its potent 

antimalarial properties, there has been an extensive scientific 

trial of artemisinin to investigate its other pharmacological 

properties. For instance, it has shown significant cytotoxic and 

antiproliferative properties against cancer cells, anti-infective 

characteristics against schistosomiases, and antiviral effects 

against certain viral particles such as hepatitis B, hepatitis 

C, human cytomegalovirus, and bovine diarrhea virus. The 

thing that is more exciting about artemisinin is its diverse 

bioactivity spectrum that includes protozoans such as 

Trypanosoma,[9] Toxoplasma gondii, Leishmania,[10] Fungi,[11] 

some Trematodes,[12] yeast, and bacteria.[13] 



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Figure 6: The impact of height on the mean artemisinin content of the 
leaves, stems, and roots of all seven Artemisia species at three different 
heights (alphabetic on the bars are ranking orders after analysis of variance 
and least significant difference) 

 

 

 
Figure 5: The variation in the average artemisinin content of the seven 
Artemisia species according to their height (alphabetic on the bars are 
ranking orders after analysis of variance and least significant difference). 
A. moorcroftiana: Artemisia moorcroftiana, A. vestita: Artemisia vestita, 

A. vulgaris: Artemisia vulgaris, A. indica: Artemisia indica, A. sieversiana: 

Artemisia sieversiana, A. roxburghiana: Artemisia roxburghiana 

 

Various methods for producing artemisinin in vitro have 

been documented, perhaps due to its possible medical uses. 

[8] Nevertheless, when it comes to synthesizing artemisinin 

on a big scale, none of these intricate systems provide a 

more feasible approach. One of the most prominent 

commercial sources of artemisinin is the herbal method of 

extracting it from A. annua, even though its production may 

be adequately achieved by genetic engineering. [15] Its yield 

from A. annua might be anywhere from 0.01% to 1.5% of 

its dry weight, according to reports. two sources: [16,17] 

Because A. annua naturally has a lower artemisinin 

concentration, it is essential to utilize other Artemisia 

species for their concealed artemisinin content. Previous 

research has shown that additional Artemisia species do, in 

fact, contain artemisinin. [6] The primary objective of this 

research was to determine the effect of altitude on the 

artemisinin content by statistically and qualitatively 

analyzing the artemisinin content in different Artemisia 

species grown at different elevations in Pakistan. 

The extraction of artemisinin from the herbal samples 

utilized toluene as one of the most widely exploited 

laboratory techniques. It is worth mentioning here that our 

findings regarding the artemisinin content may differ from 

its previously reported concentrations owing to the different 

extraction methods. We extracted 2 g of A. annua leaf powder 

in 50 mL of methanol, followed by 45-min sonication and 

3-min centrifugation at 12,000 rpm (yielding 0.03%–0.71% 

artemisinin). We then proceeded to extract 0.003% 

artemisinin from 1 g of powdered A. annua roots in 3 mL of 

toluene by letting it sit at room temperature for 30 minutes. 

Another option is to use a Soxhlet apparatus to extract 

artemisinin from 5 g of powdered A. annua leaves. We used 

200 mL of petroleum ether and heated it to 60°C for 6 hours, 

giving 0.652% artemisinin. In addition, we obtained 

0.0226%-0.785% artemisinin by refluxing 0.5 g of 

powdered A. annua leaves in 50 mL of n-hexane at 75°C for 

1 hour. As an alternative, we macerated 1 gram of fresh 

plant material in 6 milliliters of chloroform for one minute, 

which resulted in a concentration of 0.068% artemisinin.  

The amount of artemisinin in leaves is significantly affected 

by the plant's height, according to our research. While 

samples taken from 6500-foot-altitude leaves yielded 0.70% 

artemisinin per dry mass, those taken from 5400-foot-

altitude leaves yielded 0.63%. These findings largely 

corroborate previous research that has shown that height has 

a comparable effect on the concentration of artemisinin in 

the leaves. The amount of artemisinin in the leaves of A. 

roxburghiana and A. indica changed little but noticeably as a 

function of height. However, the amount of artemisinin in 

their leaves was significantly affected by their height. Figure 

6 shows that various Artemisia species were shown to 

generate greater artemisinin at height II, which was between 

5,000 and 6,000 feet. Contrary to previous reports, our data 

show that the artemisinin concentration of the leaves is 

lower at 5400 feet than at 6500 levels. 

A chemotype refers to a plant with noticeable chemical 

composition of secondary metabolites, characterized by minor 

epigenetic and genetic modifications with little or no effect 

on the morphology of the plant. The previously published 

scientific reports have highlighted prominent variations in 

artemisinin content among A. annua plants from Germany, 

China, the USA, Yugoslavia, and Vietnam. It was concluded 

that different biosynthetic precursors of artemisinin, for 



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Table 1: Seven Artemisia species collected from the three altitudes of Northern Pakistan 

Plant scientific names Height group Height (ft) Sampling point 

A. moorcroftiana I 4960 But Pul, Balakot 

A. moorcroftiana II 5804 Miandam 

A. moorcroftiana III 6511 Bhurban, Murree 

A. vestita I 1981 Yadgar Center, Basham 

A. vestita II 5363 Barhkhar Gai, Shangla 

A. vestita III 6104 Miandam 

A. vulgaris I 2829 Rawal Bridge, Basham 

A. vulgaris II 5825 Topseen, Shangla 

A. vulgaris III 8025 Mutaltan, Kalam 

A. indica I 3174 Budgran, Muzaffarabad 

A. indica II 5174 Chakar, Muzaffarabad 

A. indica III 6016 Rawalakot 

A. sieversiana I 4734 Deenoor, Gilgit 

A. sieversiana II 5901 Hasanabad, Hunza 

A. sieversiana III 7739 Kareemabad, Hunza 

A. roxburghiana var. roxburghiana I 3831 Dhery, Shangla 

A. roxburghiana var. roxburghiana II 5600 Kherabad, Miandam 

A. roxburghiana var. roxburghiana III 9480 Bahrain Road, Miandam 

A. roxburghiana var. gratae I 3831 Basham Road, Shangla 

A. roxburghiana var. gratae II 5416 Supply Stop, Rawalakot 

A. roxburghiana var. gratae III 9480 Bahreen Road, Minadam 

A. moorcroftiana: Artemisia moorcroftiana, A. vestita: Artemisia vestita, A. vulgaris: Artemisia vulgaris, A. indica: Artemisia indica, 

A. sieversiana: Artemisia sieversiana, A. roxburghiana: Artemisia roxburghiana 

 

instance, artemisinic and dihydroartemisinic acids, produced 

remarkable variability in A. annua from different geographical 

origins. These findings suggested the presence of different 

chemotypes within A. annua. In the current study, we have 

investigated the impact of altitude on the artemisinin content 

of the seven Artemisia species. These species exhibited distinct 

chemotypes, with a significant impact of their respective 

geographical conditions on their artemisinin contents. Our 

findings are quite in agreement with the previously published 

data that affirms substantial differences in artemisinin content, 

even within the same species, inferable to the climatological 

conditions.[18,19] 

As evident from the results presented in Figure 4, the height 

of various geographical locations produced a significant 

impact on the in vivo production of artemisinin in the leaves 

of five Artemisia species, namely, A. vestita, A. moorcroftiana, 

A. sieversiana, A. vulgaris, and A. roxburghiana var. 

roxburghiana. These findings affirm the existence of distinct 

chemotypes within these five Artemisia species. Our findings 

corroborate with a number of previously published reports. For 

instance, a substantial difference was observed in three key 

components – Artemisia ketone, 1,8-cineole, and camphor – of 

A. annua based on their global phytogeographic locations. 

Likewise, other studies evaluated two distinct chemotypes 

with relatively different artemisinin contents. In addition, 

researchers reported four chemotypes in Artemisia absinthium 

with a prominent difference in their essential oil composition. 

These alterations within the artemisinin content were attributed 

to natural factors such as latitude, altitude, and soil type, as 

well as the day length, flowering time, drying time, cultivation 

methods, and storage conditions. 

 

CONCLUSION 

A. annua was not the only Artemisia species found in northern 
Pakistan that contained artemisinin. Additionally, seven 
species were examined to determine the impact of habitat 
height on artemisinin production and chemotype presence or 
absence. The results showed that five Artemisia species 
exhibit chemotypes, and that Artemisia species grown at 
5000-6000 feet (height II) yield the highest leaf artemisinin 
concentration. The present research found that chemotype 
occurs among Artemisia species and that plant geographical 
habitat height impacts artemisinin production. 

 

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